Expression cassettes for transgenic expression of nucleic acids

ABSTRACT

The invention relates to expression cassettes and vectors which contain constitutive promoters of plants and to the use of these expression cassettes or vectors for transgenic expression of nucleic acid sequences, preferably selection markers, in organisms, preferably in plants. The invention further relates to transgenic plants which have been transformed with the expression cassettes or vectors, to cultures, parts or propagation material derived from these plants, and to the use of these plants for the production of food and animal feedstuffs, seed, pharmaceuticals, or fine chemicals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/755,677 filed Jan. 13, 2004, which is a national stageapplication (under 35 U.S.C. §371) of PCT/EP2002/07527 filed Jul. 5,2002, which claims benefit to German application 101 334 07.9 filed Jul.13, 2001 and German application 101 594 55.0 filed Dec. 4, 2001 andGerman application 102 075 82.4 filed Feb. 22, 2001. The entire contentsof each of these applications are hereby incorporated by referenceherein in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_List_(—)12810_(—)01171_US. The size ofthe text file is 61 KB, and the text file was created on May 20, 2011.

FIELD OF THE INVENTION

The invention relates to expression cassettes and vectors which containconstitutive promoters of plants and to the use of said expressioncassettes or vectors for transgenic expression of nucleic acidsequences, preferably selection markers, in organisms, preferably inplants. The invention further relates to transgenic plants which havebeen transformed with said expression cassettes or vectors, to cultures,parts or propagation material derived therefrom and also to the use ofsame for the production of food- and feedstuffs, seed, pharmaceuticalsor fine chemicals.

DESCRIPTION OF THE BACKGROUND

The aim of biotechnological studies on plants is the preparation ofplants having improved properties, for example to increase agriculturalproductivity. The preparation of transgenic plants is a fundamentaltechnique in plant biotechnology and thus an indispensable prerequisitefor basic research on plants in order for the preparation of plantshaving improved novel properties for agriculture, for improving thequality of foodstuffs or for the production of particular chemicals orpharmaceuticals (Dunwell J M, J Exp Bot. 2000; 51 Spec No: 487-96). Thenatural defense mechanisms of the plant, for example against pathogens,are often inadequate. The introduction of foreign genes from plants,animals or microbial sources can enhance the defense. Examples are theprotection against insects feeding on tobacco by expression of theBacillus thuringiensis endotoxin under the control of the 35 S CaMVpromoter (Vaeck et al. (1987) Nature 328:33-37) and the protection oftobacco against fungal infection by expression of a chitinase from beansunder the control of the CaMV promoter (Broglie et al. (1991) Science254:1194-1197). It is furthermore possible to achieve resistance toherbicides by introducing foreign genes, thereby optimizing thecultivation conditions and reducing crop losses (Ott K H et al. (1996) JMol Biol 263(2):359-368). The quality of the products may also beimproved. Thus it is possible, for example, to increase the shelf lifeand storability of crop products by inactivating particular maturationgenes. This was demonstrated, for example, by inactivatingpolygalacturonase in tomatoes (Hamilton A J et al. (1995) Curr TopMicrobiol Immunol 197:77-89).

A basic prerequisite for transgenic expression of particular genes inplants is the provision of plant-specific promoters. Various plantpromoters are known. It is possible to distinguish between constitutivepromoters which enable expression in various parts of a plant, which isonly slightly restricted in terms of location and time, and specificpromoters which allow expression only in particular parts or cells of aplant (e.g. root, seeds, pollen, leaves, etc.) or only at particulartimes during development. Constitutive promoters are used, for example,for expressing “selection markers”. Selection markers (e.g. antibioticor herbicidal resistance genes) permit filtering the transformationevent out of the multiplicity of untransformed but otherwise identicalindividual plants.

Constitutive promoters active in plants have been written relativelyrarely up to now. Promoters to be mentioned are the Agrobacteriumtumefaciens, TR double promoter, the promoters of the vacuolar ATPasesubunits or the promoter of a proline-rich wheat protein (WO 91/13991)and also the Ppc1 promoter Mesembryanthemum cryctallinum (Cushman et al.(1993) Plant Mol Biol 21:561-566).

The constitutive promoters which are currently the predominantly usedpromoters in plants are almost exclusively viral promoters or promotersisolated from Agrobacterium. In detail, these are the nopaline synthase(nos) promoter (Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846),the mannopine synthase (mas) promoter (Comai et al. (1990) Plant MolBiol 15 (3):373-381) and the octopine synthase (ocs) promoter (Leisnerand Gelvin (1988) Proc Natl Acad Sci USA 85(5):2553-2557) fromAgrobacterium tumefaciens and the CaMV35S promoter from cauliflowermosaic virus. The latter is the most frequently used promoter inexpression systems with ubiquitous and continuous expression (Odell etal. (1985) Nature 313:810-812; Battraw and Hall (1990) Plant Mol Biol15:527-538; Benfey et al. (1990) EMBO J. 9(69):1677-1684; U.S. Pat. No.5,612,472). However, the CaMV 35S promoter which is frequently appliedas constitutive promoter exhibits variations in its activity indifferent plants and in different tissues of the same plant (Atanassovaet al. (1998) Plant Mol Biol 37:275-85; Battraw and Hall (1990) PlantMol Biol 15:527-538; Holtorf et al. (1995) Plant Mol Biol 29:637-646;Jefferson et al. (1987) EMBO J. 6:3901-3907). A further disadvantage ofthe 35S promoter is a change in transgene expression in the case of aninfection with cauliflower mosaic virus and its typical pathogenicvariants. Thus, plants expressing the BAR gene under the control of the35S promoter are no longer resistant after infection with the viruswhich typically occurs in nature (Al-Kaff et al. (2000) NatureBiotechnology 18:995-99).

From the range of viral promoters, the sugarcane bacilliform badnavirus(ScBV) which imparts an expression pattern similar to that of CamV hasbeen described as an alternative to the CaMV 35S promoter (Schenk et al.(1999) Plant Mol Biol 39(6):1221-1230). The activity of the ScBVpromoter was analyzed in transient expression analyses using variousdicotyledonous plants, including Nicotiana tabacum and N. benthamiana,sunflower and oilseed rape, and monocotyledonous plants, here in theform of banana, corn and millet. In the transient analyses in corn, theScBV promoter-mediated expression level was comparable to that of theubiquitin promoter from corn (see below). Furthermore, the ScBVpromoter-mediated rate of expression was assayed in transgenic bananaand tobacco plants and displayed in both plant species essentiallyconstitutive expression.

Common promoters for expressing selection markers in plants areespecially the nos promoter, or else the mas promoter and ocs promoter,all of which have been isolated from Agrobacterium strains.

The use of viral sequences is often met with great reservations on thepart of the consumer. These doubts are fed not least by studies whichquestion the safety of the 35S CaMV promoter, owing to a possiblehorizontal gene transfer due to a recombination hot spot (Ho M W et al.(1999) Microbial Ecology in Health and Disease 11:194-197; Cummins J etal. (2000) Nature Biotechnology 18:363). It is therefore an aim offuture biotechnological studies on plants to replace viral geneticelements by plant regulatory elements in order to keep as closely aspossible to the plant system.

Owing to the prevailing doubts with regard to viral promoters, there areextensive efforts to replace said promoters by plant promoters. However,a promoter of plant origin, which is comparable to the viral elements,has not been described as yet.

What has been described, is a plant ubiquitin promoter from Arabidopsisthaliana (Callis et al. (1990) J Biol Chem 265:12486-12493; Holtorf S etal. (1995) Plant Mol Biol 29:637-747). Contrary to the findings in thearticles mentioned, some studies revealed that the Arabidopsis ubiquitinpromoter is unsuitable for expressing selection marker genes and that,for this reason, its general applicability must be called into question(see comparative examples 1 and 3).

The expression pattern mediated by the corn ubiquitin promoter has beendescribed for the Ubi-1 and Ubi-2 promoters from corn (Christensen etal. (1992) Plant Mol Biol 18(4):675-689). While the Ubi-1 promoter hasgood expression activity in corn and other monocotyledonous plants, itexhibits in dicotyledonous tobacco plants only 10% of the activity whichhad been achieved in comparable experiments using the viral 35Spromoter. It was furthermore shown that the corn Ubi-1 promoter issuitable for over expression of genes in monocotyledonous plant systemsand, in addition, is sufficiently strong in order to mediate aherbicidal resistance via the expression of selection markers(Christensen and Quail (1996) Transgenic Res 5(3):213-218). The Ubi-1promoter proved unsuitable for dicotyledonous expression systems.

A comparison of the organ specificity and strength of variousconstitutive promoters was carried out by Holtorf (Holtorf et al. (1995)Plant Mol Biol 29(4):637-646) on the basis of stably transformedArabidopsis plants. The study comprised, inter alia, the CaMV35Spromoter, the leaf-specific thionine promoter from barley and theArabidopsis ubiquitin promoter (UBQ1). The CaMV35S promoter exhibitedthe highest rate of expression. On the basis of using an additionaltranslational enhancer (TMV omega element), it was possible to increasethe rate of expression of the promoter by a factor of two to three withunchanged organ specificity. The leaf-specific thionine promoter frombarley was inactive in the majority of transformed lines, while the UBQ1promoter from Arabidopsis resulted in medium rates of expression.

McElroy and colleagues reported a construct for transformingmonocotyledonous plants, which is based on the rice actin 1 (Act1)promoter (McElroy et al. (1991) Mol Gen Genet. 231:150-160). Overall, itwas concluded from the afore-described studies that the Act1promoter-based expression vectors are suitable for controlling asufficiently strong and constitutive expression of foreign DNA intransformed cells of monocotyledonous plants.

Another constitutive promoter which has been described is the promoterof an S-adenosyl-L-methionine synthetase (WO 00/37662). A disadvantagehere is especially a dependence of the strength of expression on themethionine concentration (see WO 00/37662; FIG. 7).

WO 99/31258 describes chimeric constitutive plant promoters which arecomposed of various elements of various promoters with complementaryexpression patterns so that combination of individual tissuespecificities additively results in a constitutive expression pattern.

Ferredoxin NADPH oxidoreductase (FNR) is a protein of the electrontransport chain and reduces NADP+ to NADPH. Experiments in spinach usingthe spinach FNR promoter fused to the GUS gene hint at a light-inducibleelement in the FNR promoter (Oelmüller et al. (1993) Mol. Gen. Genet.237:261-72). Owing to its function, a strictly leaf-specific expressionpattern would have been expected for the promoter. Owing to thetissue-dependent expression pattern, the promoter would be poorly suitedto expressing selection markers. Here, a selection in all tissue parts,if possible, is required in order to ensure efficient selection.

Owing to its function during photosynthesis, the promoter of the triosephosphate translocator (TPT) should be mainly leaf-specific. The cDNAsfrom potato (Schulz et al. (1993) Mol Gen Genet. 238:357-61),cauliflower (Fischer et al. (1997) Plant Cell 9:453-62), oilseed rape(WO 97/25346) and corn Kammerer B (1998) The Plant Cell 10:105-117) havebeen described. Kammerer et al. demonstrate that TPT mRNA expression incorn is strong in the leaves and the stamen. In contrast, no expressionwas observed in the stem or in the roots. Owing to the tissue-dependentexpression pattern, the promoter would be poorly suited to expressingselection markers. Here, a selection in all tissue parts, if possible,is required in order to ensure efficient selection.

The “constitutive” promoters described in the prior art have one or moreof the following disadvantages:

1. Inadequate homogeneity of expression:

The known “constitutive” promoters frequently display a different levelof expression, depending on the type of tissue or cell. Moreover, theexpression property is often highly dependent on the site of insertioninto the host genome. As a consequence of this, the effects to beobtained by heterologous expression cannot be achieved to the sameextent homogeneously in the plant. Under or over dosages may occur. Thismay have an adverse effect on plant growth or plant value.

2. Inadequate time profile:

The “constitutive” promoters known in the prior art often exhibit anonconsistent activity during the development of a tissue. As a result,it is not possible, for example, to achieve desirable effects (such asselection) in the early phase of somatic embryogenesis which would beadvantageous, especially here, due to the sensitivity of the embryo toin vitro conditions and stress factors.

3. Inadequate applicability to many plant species:

The “constitutive” promoters described in the prior art are often notactive in the same way in all species.

4. If a plurality of expression cassettes with in each case the same“constitutive” promoter are present in an organism, interactions betweensaid expression cassettes and even switching-off (gene silencing) ofindividual expression cassettes may occur (Mette et al. (1999) EMBO J.18:241-248).

5. Promoters of viral origin may be influenced by virus infections ofthe transgenic plant and may then no longer express the desired property(Al-Kaff et al. (2000) Nature Biotechnology 18:995-99).

6. The public acceptance toward the use of promoters and elements fromplant systems is higher than for viral systems.

7. The number of promoters suitable for expressing selection markers inplants is low and said promoters are usually of viral or bacterialorigin.

8. Pollen/anther expression: The promoters mentioned (such as, forexample, 35S CaMV) exhibit strong activity in the pollen or in theanthers. This may have disadvantageous effects on the environment. Thus,unspecific expression of Bacillus thuringiensis endotoxins resulted notonly in the desired effect on feeding insects due to expression in theroot but also, due to expression in the pollen, in considerable damagein the population of the Monarch butterfly which feeds predominantly onthe pollen (Losey J E et al. (1999) Nature 399, 214).

An ideal constitutive promoter should have as many of the followingproperties as possible: (a) a gene expression which is as homogeneous aspossible with regard to location and time, i.e. an expression in as manycell types or tissues of an organism as possible during the variousphases of the developmental cycle. Furthermore, an efficient selectionin differentiated cells (various callus phases) from a tissue cultureand other developmental stages suitable for tissue culture is desired,(b) an applicability to various plant species, which is as broad aspossible, and applicability to species in which it is not possible toachieve any expression using the “constitutive” promoters known to date,(c) to combine a plurality of transgenes in one plant, it is desirableto carry out a plurality of transformations in succession or to useconstructs with a plurality of promoter cassettes, but withoutgenerating silencing effects due to the multiple use of identicalregulatory sequences, (d) a plant origin in order to avoid problems ofacceptance by the consumer and possible problems of future approval, and(e) secondary activities of a promoter in the anthers/pollen areundesirable, for example in order to avoid environmental damage, asdiscussed herein.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides new toolsand methods for the expression of nucleic acids.

One embodiment of the invention is directed to expression cassettes fortransgenic expression of nucleic acids, comprising a promoter containingthe sequence of SEQ ID NO: 1, 2 or 3 which possesses a promoteractivity, or a fragment, functional equivalent or equivalent fragmentthereof which possesses said promoter activity, wherein said promoter orsaid fragment is functionally linked to a nucleic acid sequence to beexpressed transgenically. The equivalent fragment may comprise asequence that contains the sequence of SEQ ID NO: 4 or SEQ ID NO: 27.The nucleic acid sequence to be expressed may be functionally linked toone or more further genetic control sequences, or the expressioncassette contains one or more additional functional elements. Transgenicexpression with cassettes of the invention enables expression of aprotein encoded by said nucleic acid sequence, or expression of a senseor antisense RNA encoded by said nucleic acid sequence. Preferrednucleic acid sequences to be expressed transgenically include, but arenot limited to nucleic acids coding for selection markers, reportergenes, cellulases, chitinases, glucanases, ribosome-inactivatingproteins, lysozymes, Bacillus thuringiensis endotoxin, α-amylaseinhibitor, protease inhibitors, lectins, RNAases, ribozymes, acetyl-CoAcarboxylases, phytases, 2S albumin from Bertholletia excelsa, antifreezeproteins, trehalose phosphate synthase, trehalose phosphate phosphatase,trehalase, DREB1A factor, farnesyl transferases, ferritin, oxalateoxidase, calcium-dependent protein kinases, calcineurins, glutamatedehydrogenases, N-hydroxylating multifunctional cytochrome P450,transcriptional activator CBF1, phytoene desaturases,polygalacturonases, flavonoid 3′-hydroxylases, dihydroflavanol4-reductases, chalcone isomerases, chalcone synthases, flavanone3-beta-hydroxylases, flavone synthase II, branching enzyme Q, starchbranching enzyme or combinations thereof. In addition, nucleic acidsequences to be expressed transgenically may also include the nucleicacid sequences with GenBank accession numbers U77378, AF306348, A19451,L25042, S78423, U32624, X78815, AJO02399, AFO078796, ABO44391, AJ222980,X14074, AB045593, AFO17451, AF276302, ABO61022, X72592, AB045592, orAR123356.

Preferably, the nucleic acid sequence to be expressed transgenically isa positive selection marker, a negative selection marker, a factor thatgives a growth advantage, or a combinations thereof. Preferred positiveor negative selection marker include proteins that impart a resistanceto antibiotics, metabolism inhibitors, herbicides, biocides, proteinsthat impart a resistance to phosphinothricin, glyphosate, bromoxynil,dalapon, 2-deoxyglucose 6-phosphate, tetracyclines, ampicillin,kanamycin, G418, neomycin, paromomycin, bleomycin, zeocin, hygromycin,chloramphenicol, sulfonyl urea herbicides, imidazolinone herbicides, orcombinations thereof.

Preferred selection markers include phosphinothricin acetyltransferases,5-enolpyruvylshikimate 3-phosphate synthases, glyphosateoxidoreductases, dehalogenases, nitrilases, neomycinphosphotransferases, DOG^(R)1 genes, acetolactate synthases, hygromycinphosphotransferases, chloramphenicol acetyltransferases, streptomycinadenylyltransferases, β-lactamases, tetA genes, tetR genes, isopentenyltransferases, thymidine kinases, diphtheria toxin A, cytosine deaminase(codA), cytochrome P450, haloalkanedehalogenases, iaaH gene, tms2 gene,β-glucuronidases, mannose 6-phosphate isomerases, UDP-galactose4-epimerases and combinations thereof.

Positive or negative selection markers may be encoded by a nucleic acidthat contains the sequence of SEQ ID NO: 5 or 6; or the sequence ofGenBank Acc. No.: X17220, X05822, M22827, X65195, AJ028212, X17220,X05822, M22827, X65195, AJ028212, X63374, M10947, AX022822, AX022820,E01313, J03196, AF080390, AF234316, AF080389, AF234315, AF234314,U00004, NC001140, X51514, AB049823, AF094326, X07645, X07644, A19547,A19546, A19545, 105376, 105373, X74325, AF294981, AF234301, AF234300,AF234299, AF234298, AF354046, AF354045, X65876, X51366, AJ278607,L36849, AB025109, or AL133315.

The nucleic acid sequence to be expressed from the expression cassettemay be functionally linked to one or more further genetic controlsequences, and/or one or more additional functional elements.

Another embodiment of the invention is directed to vectors comprisingthe expression cassette of the invention.

Another embodiment of the invention is directed to transgenic organismstransformed with vectors of the invention. Preferred transgenicorganisms include bacteria, yeasts, fungi, animal and plant organisms.Preferred plant organisms include Arabidopsis, tomato, tobacco,potatoes, corn, oilseed rape, wheat, barley, sunflowers, millet, beet,rye, oats, sugarbeet, bean plants and soyabean.

Another embodiment of the invention is directed to a cell culture, plantor transgenic propagation material, derived from a transgenic organismof the invention.

Another embodiment of the invention is directed to methods fortransgenic expression of nucleic acids comprising transgenicallyexpressing a nucleic acid sequence which is functionally linked to apromoter containing the sequence of SEQ ID NO: 1, 2 or 3 and has apromoter activity; or a fragment, functional equivalent or equivalentfragment thereof which possesses the promoter activity. Functionallyequivalent fragments may contain a sequence such as, but not limited tothe sequences of SEQ ID NO: 4 and 27. Preferably, the nucleic acidsequence to be expressed is functionally linked to one or more furthergenetic control sequences, and one or more additional functionalelements, and transgenically enables the expression of a protein encodedby said nucleic acid sequence, or the expression of a sense or antisenseRNA encoded by said nucleic acid sequence.

Another embodiment of the invention is directed to methods for selectingtransformed organisms comprising introducing a nucleic acid sequencecoding for a selection marker to said organisms, wherein said nucleicacid sequence is functionally and transgenically linked to a promoteraccording to SEQ ID NO: 1, 2 or 3, which possesses a promoter activity;or a fragment, functional equivalent or equivalent fragment thereofwhich possesses said promoter activity; selecting organisms expressingsaid selection marker; and isolating selected organisms. Preferredselection markers include nucleic acid sequences with GenBank accessionnumbers U77378, AF306348, A19451, L25042, S78423, U32624, X78815,AJO02399, AFO78796, ABO44391, AJ222980, X14074, AB045593, AFO17451,AF276302, ABO61022, X72592, AB045592, or AR123356, positive selectionmarkers, negative selection markers, factors which give a growthadvantage, proteins which impart a resistance to antibiotics, metabolisminhibitors, herbicides, biocides, phosphinothricin acetyltransferases,5-enolpyruvylshikimate 3-phosphate synthases, glyphosateoxidoreductases, dehalogenases, nitrilases, neomycinphosphotransferases, DOG^(R)1 genes, acetolactate synthases, hygromycinphosphotransferases, chloramphenicol acetyltransferases, streptomycinadenylyltransferases, β-lactamases, tetA genes, tetR genes, isopentenyltransferases, thymidine kinases, diphtheria toxin A, cytosine deaminase(codA), cytochrome P450, haloalkanedehalogenases, iaaH gene, tms2 gene,β-glucuronidases, mannose 6-phosphate isomerases, UDP-galactose4-epimerases and combinations thereof.

Another embodiment of the invention is directed to methods for theproduction of a foodstuff, a feedstuff, a seed, a pharmaceutical or afine chemical comprising propagating the transgenic organism of theinvention or cell cultures, parts or transgenic propagation materialderived therefrom. Optionally, methods further comprise growing thetransgenic organism and isolating the foodstuff, feedstuff, seed,pharmaceutical or fine chemical. Preferred fine chemicals include, butare not limited to enzymes, vitamins, amino acids, sugars, saturated orunsaturated fatty acids, natural or synthetic flavorings, aromatizingsubstances and colorants. Preferred pharmaceuticals include, but are notlimited to an antibody, enzyme or pharmaceutically active protein.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. The TPT and the FNR promoters show a comparable expressionpattern in green tissue and in flowers of tobacco and potato. (a) A:Potato leaves. B: Tobacco petioles. (b) C: Tobacco stems. D: Tobaccointernodia. (c) E: Tobacco flower.

FIG. 2. The TPT promoter and the FNR promoter show a differentexpression pattern in vegetative and germinative storage tissue oftobacco and potato. (a) A: Tobacco seeds. B: Potato tubers. (b) C:Tobacco seedlings.

FIG. 3. Expression cassettes for the expression of kanamycin-resistance(nptII) and phosphinothricin-resistance (pat) markers.

FIG. 4. Regeneration of transformed tobacco plumulae under kanamycinselection pressure. A: transformation with an FNR promoter-nptIIconstruct. B; transformation with a TPT promoter-nptII construct.

FIG. 5. Germination of transformed tobacco plants from transgenictobacco seeds under phosphinothricin selection pressure. A: transformedwith an FNR promoter-pat construct. B: transformed with a TPTpromoter-pat construct. C: control with untransformed tobacco seeds.

DESCRIPTION OF THE INVENTION

The present invention is directed to providing regulatory sequences ofplants, which fulfill as many of the herein mentioned properties aspossible and which mediate especially a ubiquitous anddevelopment-independent (constitutive) expression of a nucleic acidsequence to be expressed which preferably codes for a selection marker.Despite various plant promoters for which a constitutive expression atleast in individual species is claimed, no promoter having the desiredproperties listed herein has been described up to now. The invention isfurther directed to the identification of appropriate promoters.

We found that this is achieved by providing expression cassettes basedon the promoters of a putative ferredoxin gene (pFD “putativeferredoxin” herein) from Arabidopsis thaliana, of the ferredoxin NADPHoxidoreductase (FNR herein) gene from Arabidopsis thaliana and of thetriose phosphate translocator (TPT) gene from Arabidopsis thaliana:

Promoter of a Putative Ferredoxin (pFD) from Arabidopsis thaliana:

During analysis of the Arabidopsis genome, the ORF of a putativeferredoxin gene was identified. The isolated 836 bp 5′-flanking sequencefused to the Glucuronidase gene surprisingly exhibited a constitutiveexpression pattern in transgenic tobacco. The sequence corresponds to asequence section on Arabidopsis thaliana chromosome 4, as it has beendeposited at GenBank under Acc. No. Z97337 (Version Z97337.2; base pair85117 to 85952; the gene starting from bp 85953 is annotated “strongsimilarity to ferredoxin [2Fe-2S] I, Nostoc muscorum”). (The gene is notto be confused with the A. thaliana gene for preferredoxin annotatedunder GenBank Acc.—NoAcc. No: X51370; Vorst O et al. (1990) Plant MolBiol 14(4):491-499).

Only a weak activity was detected in the anthers/pollen of the closedflower buds and no activity whatsoever was detected in mature flowers.Contrary to the reservations, derived from the findings in theliterature, toward a suitability of the promoter for effectiveexpression of selection markers (for example, owing to the suspectedleaf specificity or function in the photosynthetic electron transport),it was possible to demonstrate a highly efficient selection bycombination with, for example, the homolog resistance gene (nptII).

2.) Ferredoxin NADPH Oxidoreductase (FNR) Promoter from Arabidopsisthaliana:

Starting from the information on FNR-encoding cDNA from N. tabacum(GenBank Acc. No.: Y14032) the Arabidopis data base was screened for ahomologous gene. Primers were synthesized according to said sequenceinformation. The promoter amplified via PCR from Arabidopsis thalianagenomic DNA (635 bp), of which a leaf-specific expression was expected,exhibited in transgenic tobacco plants a surprisingly ubiquitous andinsertion site-independent expression.

The promoter sequence partly corresponds to a sequence section onArabidopsis thaliana chromosome 5, as it is deposited at GenBank underAcc. No. AB011474 (Version AB011474.1 from 12.27.2000; base pair 70127to 69493; the gene starting at by 69492 is annotated with“ferredoxin-NADP+reductase”).

No activity was detected in the pollen. Contrary to the reservations,derived from the findings in the literature, toward a suitability of thepromoter for effective expression of selection markers (for example,owing to the suspected leaf specificity or function in thephotosynthetic electron transport), it was possible to demonstrate ahighly efficient selection by combination with, for example, thephosphinothricin resistance gene (bar/pat).

The nondetectable activity of the FNR promoter in seeds allows a use forthe expression of genes whose gene products are desired in other partsof the plant and are unwanted in the seeds. For example, pests can berepelled by expressing appropriate toxins such as, for example, Bacillusthuringiensis crystal proteins. Thus it is possible to achieve inpotatoes expression in the plant organs above the ground (and thus, forexample, a repulsion of pests such as the potato beetle) withoutsimultaneous expression in the tuber which is used as food or animalfeed, and this could increase the suitability and acceptance.

3.) Triose Phosphate Translocator (TPT) Promoter from Arabidopsisthaliana:

A 2038 bp PCR fragment was amplified, starting from Arabidopsis GenBankdata of chromosome V, clone MCL19. The promoter sequence partlycorresponds to a sequence section on Arabidopsis thaliana chromosome 5,as it is deposited with GenBank under Ace. No. AB006698 (VersionAB006698.1 from Dec. 27, 2000; base pair 53242 to 55281; the genestarting at by 55282 is annotated with “phosphate/triose-phosphatetranslocator”).

Surprisingly, transgenic tobacco plants exhibited not only a highactivity in numerous parts of the plant. No activity was detected in thepollen. Contrary to the reservations, derived from the findings in theliterature, toward a suitability of the promoter for effectiveexpression of selection markers (for example, owing to the suspectedleaf specificity), it was possible to demonstrate a highly efficientselection by combination with, for example, the phosphinothricinresistance gene (bar/pat).

The ubiquitous expression pattern, but especially also the ability ofthe TPT promoter regarding the expression of selection markers, comes asa great surprise for the skilled worker, since the triosephosphatetranslocator is responsible for the exchange of C3 sugar phosphatesbetween the cytosol and the plastids in photosynthetic leaves. The TPTis located in the inner chloroplast membrane. Colorless plastidstypically contain a hexose transporter via which C6-sugar phosphates areexchanged. It is not to be expected that such genes are active in theearly callus and embryogenesis stages (Stitt (1997) Plant Metabolism,2nd ed., Dennis eds. Longman Press, Harlow, U K, 382-400).

The pFD, FNR and TPT promoters proved to be sufficiently strong in orderto express nucleic acid sequences, in particular selection marker genes,successfully. Furthermore, various deletion variants of theabovementioned promoters, in particular a truncated variant of the pFDpromoter (699 bp) and of the TPT promoter (1318 bp), proved suitable forensuring the expression of, for example, selection markers such as thehomolog resistance (nptII).

Furthermore, the Arabidopsis thaliana ubiquitin promoter (Holtorf et al.(1995) Plant Mol Biol 29:637-646) and the squalene synthase promoter(Kribii et al. (1997) Eur J Biochem 249:61-69) were studied within theframework of the studies mentioned, both of which, however, weresurprisingly unsuitable for mediating selection marker gene expressionalthough the literature data of the ubiquitin promoters frommonocotyledons (see above) had led to the assumption that in particularthe ubiquitin promoter of a dicotyledonous plant should have worked as apromoter of a selection marker system (see comparative examples 1 and3). A similar statement applies to the squalene synthase promoter whosecharacterization had led to the expectation that it would be possible toachieve sufficiently high rates of expression for the successful controlof a selection marker gene (Del Arco and Boronat (1999) 4th EuropeanSymposium on Plant Isoprenoids, 21.-Dec. 23, 1999, Barcelona, Spain)(see comparative examples 2 and 3).

The present invention therefore relates firstly to expression cassettesfor transgenic expression of nucleic acids, comprising: a) a promoteraccording to SEQ ID No: 1, 2 or 3, b) a functional equivalent orequivalent fragment of a), which essentially possesses the same promoteractivity as a), a) or b) being functionally linked to a nucleic acidsequence to be expressed transgenically.

The invention further relates to methods for transgenic expression ofnucleic acids, wherein a nucleic acid sequence which is functionallylinked to a) a promoter according to SEQ ID NO: 1, 2 or 3 or b) afunctional equivalent or equivalent fragment of a) which essentiallypossesses the same promoter activities as a), is expressedtransgenically.

Expression comprises transcription of the nucleic acid sequence to beexpressed transgenically but may also include, in the case of an openreading frame in sense orientation, translation of the transcribed RNAof the nucleic acid sequence to be expressed transgenically into acorresponding polypeptide.

An expression cassette for transgenic expression of nucleic acids or amethod for transgenic expression of nucleic acids comprises all thoseconstructions produced by genetic methods or methods in which either a)a promoter according to SEQ ID No: 1, 2 or 3 or a functional equivalentor equivalent fragment thereof, or b) the nucleic acid sequence to beexpressed, or c) (a) and (b) are not present in their natural geneticenvironment (i.e. at their natural chromosomal locus) or have beenmodified by genetic methods, and said modification may be, by way ofexample, a substitution, addition, deletion, inversion or insertion ofone or more nucleotide residues.

The expression cassettes of the invention, vectors derived from them orthe methods of the invention may comprise functional equivalents of thepromoter sequences described under SEQ ID No: 1, 2 or 3. Functionallyequivalent sequences also comprise all those sequences which are derivedfrom the complementary counter strand of the sequences defined by SEQ IDNO: 1, 2 or 3 and which have essentially the same promoter activity.

Functional equivalents with respect to the promoters of the inventionmeans in particular natural or artificial mutations of the promotersequences described under SEQ ID No: 1, 2 or 3 and of the homologsthereof from other plant genera and species, which furthermore haveessentially the same promoter activity.

A promoter activity is essentially referred to as identical, if thetranscription of a particular gene to be expressed under the control ofa particular promoter derived from SEQ ID NO: 1, 2 or 3 under otherwiseunchanged conditions has a location within the plant, which is at least50%, preferably at least 70%, particularly preferably at least 90%, veryparticularly preferably at least 95%, congruent with that of acomparative expression obtained using a promoter described by SEQ ID NO:1, 2 or 3. In this connection, the expression level may deviate bothdownward and upward in comparison to a comparative value. In thisconnection, preference is given to those sequences whose expressionlevel, measured on the basis of the transcribed mRNA or the subsequentlytranslated protein, differs quantitatively by not more than 50%,preferably 25%, particularly preferably 10%, from a comparative valueobtained using a promoter described by SEQ ID NO: 1, 2 or 3, underotherwise unchanged conditions. Particular preference is given to thosesequences whose expression level, measured on the basis of thetranscribed mRNA or of the subsequently translated protein, isquantitatively more than 50%, preferably 100%, particularly preferably500%, very particularly preferably 1000%, higher than a comparativevalue obtained with the promoter described by SEQ ID NO: 1, 2 or 3,under otherwise unchanged conditions. The comparative value ispreferably the expression level of the natural mRNA of the particulargene or of the natural gene product. A further preferred comparativevalue is the expression level obtained using a random but particularnucleic acid sequence, preferably those nucleic acid sequences whichcode for readily quantifiable proteins. In this connection, veryparticular preference is given to reporter proteins (Schenborn E,Groskreutz D. Mol. Biotechnol. 1999; 13(1):29-44) such as the greenfluorescence protein (GFP) (Chui W L et al., Curr Biol 1996, 6:325-330;Leffel S M et al., Biotechniques. 23(5):912-8, 1997), chloramphenicoltransferase, a luciferase (Millar et al., Plant Mol Biol Rep 199210:324-414) or β-galactosidase, and very particular preference is givento .beta.-glucuronidase (Jefferson et al. (1987) EMBO J. 6:3901-3907).

Otherwise unchanged conditions means the expression initiated by one ofthe expression cassettes to be compared is not modified by a combinationwith additional genetic control sequences, for example enhancersequences. Unchanged conditions further means that all basic conditionssuch as, for example, plant species, developmental stage of the plants,growing conditions, assay conditions (such as buffer, temperature,substrates, etc.) are kept identical between the expressions to becompared.

Mutations comprise substitutions, additions, deletions, inversions orinsertions of one or more nucleotide residues. Thus, for example, thepresent invention also includes those nucleotide sequences which areobtained by modification of a promoter of SEQ ID NO: 1, 2 or 3. The aimof such a modification may be the further narrowing down of the sequencecomprised therein or else, for example, the introduction of furtherrestriction enzyme cleavage sites, the removal of excess DNA or theaddition of further sequences, for example of further regulatorysequences.

Where insertions, deletions or substitutions such as, for example,transitions and transversions are suitable, techniques known per se,such as in vitro mutagenesis, “primer repair”, restriction or ligation,may be used. Manipulations such as, for example, restriction,chewing-back or filling-in of protruding ends to give blunt ends canprovide complementary fragment ends for ligation. Similar results can beobtained using the polymerase chain reaction (PCR) using specificoligonucleotide primers.

Homology between two nucleic acids means the identity of the nucleicacid sequence over the in each case entire length of the sequence, whichis calculated by comparison with the aid of the GAP program algorithm(Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), with the parameters set as follows:

Gap Weight: 12 Length Weight: 4 Average Match: 2.912 Average Mismatch:−2.003

By way of example, a sequence which is at least 50% homologous at thenucleic acid level with the sequence SEQ ID NO: 1, 2 or 3 means asequence which is at least 50% homologous when compared to the sequenceSEQ ID NO. 1, 2 or 3 according to the above program algorithm using theabove set of parameters.

Functional homologs to the abovementioned promoters for use in theexpression cassettes of the invention preferably include those sequenceswhich, are at least 50%, preferably 70%, preferentially at least 80%,particularly preferably at least 90%, very particularly preferably atleast 95%, most preferably 99%, homologous over a length of at least 100base pairs, preferably at least 200 base pairs, particularly preferablyat least 300 base pairs, very particularly preferably at least 400 basepairs and most preferably of at least 500 base pairs.

Further examples of the promoter sequences employed in the expressioncassettes or vectors of the invention can readily be found, for example,in various organisms whose genomic sequence is known, such as, forexample, Arabidopsis thaliana, Brassica napus, Nicotiana tabacum,Solanum tuberosum, Helianthium anuus, Linum sativum by comparinghomologies in databases.

Functional equivalents further means DNA sequences which hybridize understandard conditions with the nucleic acid sequence coding for a promoteraccording to SEQ ID NO:1, 2 or 3 or with the nucleic acid sequencescomplementary to it and which have essentially the same properties.Standard hybridization conditions has a broad meaning and means bothstringent and less stringent hybridization conditions. Suchhybridization conditions are described, inter alia, in Sambrook J,Fritsch E F, Maniatis T et al., in Molecular Cloning—A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pp.9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6.

The conditions during the washing step may be selected by way of examplefrom the range of conditions limited by those of low stringency (withapproximately 2.times.SSC at 50° C.) and those with high stringency(with approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC:0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition, the temperaturemay be raised during the washing step from low stringency conditions atroom temperature, approximately 22° C., to higher stringency conditionsat approximately 65° C. Both parameters, salt concentration andtemperature, may be varied simultaneously, and it is also possible tokeep one of the two parameters constant and to vary only the other one.Denaturing agents such as, for example, formamide or SDS may also beemployed during hybridization. In the presence of 50% formamide,hybridization is preferably carried out at 42° C. Some exemplaryconditions for hybridization and washing are listed below:

(1) Hybridization conditions with, for example,

a) 4×SSC at 65° C., or

b) 6×SSC, 0.5% SDS, 10.mu.g/ml denatured, fragmented salmon sperm DNA at65° C., orc) 4×SSC, 50% formamide, at 42° C., ord) 6×SSC, 0.5% SDS, 10.mu.g/ml denatured, fragmented salmon sperm-DNA,50% formamide at 42° C., ore) 2× or 4×SSC at 50° C. (low stringency condition), orf) 2× or 4×SSC, 30 to 40% formamide at 42° C. (low stringencycondition).

g) 6×SSC at 45° C., or,

h) 50% formamide, 4×SSC at 42° C., ori) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 75 mM NaCl,75 mM sodium citrate at 42° C., orj) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and 7% SDS.

(2) Washing steps with, for example:

a) 0.1 C SSC at 65° C., or b) 0.1×SSC, 0.5% SDS at 68° C., or

c) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or

d) 0.2×SSC, 0.1% SDS at 42° C., or

e) 2×SSC at 65° C. (low stringency condition), orf) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.

Methods for preparing functional equivalents of the invention preferablycomprise introducing mutations into a promoter of SEQ ID NO: 1, 2 or 3.A mutagenesis may be random and the mutagenized sequences aresubsequently screened with respect to their properties according to atrial-by-error procedure. Examples of particularly advantageousselection criteria are an increased resistance to a selection marker andthe level of the resulting expression of the introduced nucleic acidsequence.

As an alternative, it is possible to delete non-essential sequences of apromoter of the invention without substantially impairing saidproperties. Such deletion variants are functionally equivalent fragmentsof the promoters described by SEQ ID NO: 1, 2 or 3. Examples of suchdeletion mutants or functionally equivalent fragments, which may bementioned, are the truncated pFD promoter sequence (pFDs) according toSEQ ID NO: 4 and the truncated TPT promoter sequence according to SEQ IDNO: 27 which, as functionally equivalent parts of their respectivesource promoters, are expressly included.

The narrowing-down of the promoter sequence to particular essentialregulatory regions may also be carried out with the aid of searchroutines for searching for promoter elements. Particular promoterelements are often present in increased numbers in the regions relevantfor promoter activity. Said analysis may be carried out, for example, bycomputer programs such as the program PLACE (“Plant Cis-actingRegulatory DNA Elements”) (K. Higo et al., (1999) Nucleic Acids Research27:1, 297-300) or the BIOBASE data bank “Transfac” (BiologischeDatenbanken GmbH, Brunswick).

Methods for mutagenizing nucleic acid sequences are known to the skilledworker and include, by way of example, the use of oligonucleotideshaving one or more mutations in comparison with the region to be mutated(for example, within the framework of a site-specific mutagenesis).Typically, primers with from approximately 15 to approximately 75nucleotides or more are employed, preferably from approx. 10 to approx.25 or more nucleotide residues being located on both sites of thesequence to be modified. Details and the procedure of said mutagenesismethods are familiar to the skilled worker (Kunkel et al., MethodsEnzymol, 154:367-382, 1987; Tomic et al. (1990) Nucl Acids Res 12:1656;Upender, Raj, Weir (1995) Biotechniques 18(1):29-30; U.S. Pat. No.4,237,224). A mutagenesis may also be carried out by treating, forexample, vectors comprising one of the nucleic acid sequences of theinvention with mutagenizing agents such as hydroxylamine.

The nucleic acid sequences which are comprised in the expressioncassettes of the invention and which are to be expressed transgenicallymay be functionally linked to further genetic control sequences, inaddition to one of the promoters of the invention.

A functional linkage means, for example, the sequential arrangement of apromoter, of the nucleic acid sequence to be expressed transgenicallyand, where appropriate, of further regulatory elements such as, forexample, a terminator in such a way that each of the regulatory elementscan carry out its function in the transgenic expression of said nucleicacid sequence, depending on the arrangement of the nucleic acidsequences with respect to sense or antisense RNA. This does notabsolutely necessitate a direct linkage in the chemical sense. Geneticcontrol sequences such as, for example, enhancer sequences may exerttheir function on the target sequence also from relatively distantpositions or even from other DNA molecules. Preference is given toarrangements in which the nucleic acid sequence to be expressedtransgenically is positioned downstream of the sequence functioning aspromoter so that both sequences are covalently linked to one another.The distance between the promoter sequence and the nucleic acid sequenceto be expressed transgenically is preferably less than 200 base pairs,particularly preferably less than 100 base pairs and very particularlypreferably less than 50 base pairs.

A functional linkage may be prepared by means of common recombinationand cloning techniques, as are described, for example, in T. Maniatis,E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and in T.J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984)and in Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley Interscience (1987). It is alsopossible to position further sequences between said two sequences, whichhave, for example, the function of a linker with particular restrictionenzyme cleavage sites or of a signal peptide. Likewise, the insertion ofsequences may lead to the expression of fusion proteins.

The term “genetic control sequences” has a broad meaning and means allthose sequences which influence the generation or function of theexpression cassette of the invention. For example, genetic controlsequences modify transcription and translation in prokaryotic oreukaryotic organisms. The expression cassettes of the inventionpreferably comprise 5′-upstream of the particular nucleic acid sequenceto be expressed transgenically one of the promoters of the invention and3′-downstream a terminator sequence as an additional genetic controlsequence and also, where appropriate, further common regulatory elementswhich are in each case functionally linked to the nucleic acid sequenceto be expressed transgenically.

Genetic control sequences also include further promoters, promoterelements or minimal promoters which may modify theexpression-controlling properties. Thus, for example, genetic controlsequences can effect tissue-specific expression additionally dependingon particular stress factors. Corresponding elements have beendescribed, for example, for water stress, abscisic acid (Lam E and ChuaN H (1991) J Biol Chem 266(26): 17131-17135) and heat stress (Schoffl Fet al., (1989) Molecular & General Genetics 217(2-3):246-53).

Further promoters which make possible expression in further planttissues or in other organisms such as, for example, in E. coli bacteriamay furthermore be functionally linked to the nucleic acid sequence tobe expressed. Suitable plant promoters are in principle all of theabove-described promoters. It is conceivable, for example, that aparticular nucleic acid sequence is transcribed as sense RNA via onepromoter (for example one of the promoters of the invention) in oneplant tissue and is translated into the corresponding protein, while thesame nucleic acid sequence is transcribed to antisense RNA via anotherpromoter having a different specificity in another tissue and thecorresponding protein is down-regulated. This may be carried out via anexpression cassette of the invention by positioning the one promoterupstream of the nucleic acid sequence to be expressed transgenically andthe other promoter downstream of said sequence.

Genetic control sequences furthermore also include the 5′-untranslatedregion, introns or the noncoding 3′-region of genes, preferably of thepFD, FNR or TPT-genes. It has been demonstrated that these genes mayhave a substantial function in the regulation of gene expression. Thusit was shown that 5′-untranslated sequences can enhance transientexpression of heterologous genes. They may furthermore promote tissuespecificity (Rouster J et al. (1998) Plant J. 15:435-440). Conversely,the 5′-untranslated region of the opaque-2 gene suppresses expression. Adeletion of the corresponding region leads to an increase in geneactivity (Lohmer S et al. (1993) Plant Cell 5:65-73). The nucleic acidsequence indicated under SEQ ID NO:1, 2 or 3 contains the pFD, FNR orTPT-gene section which represents the promoter and the 5′-untranslatedregion up to the ATG start codon of the respective protein.

McElroy and colleagues (McElroy et al. (1991) Mol Gen Genet231(1):150-160) reported a construct for transforming monocotyledonousplants, which is based on the rice actin 1 (Act1) promoter. The use ofthe Act1 intron in combination with the 35S promoter leads in transgenicrice cells to a ten times higher rate of expression compared to theisolated 35S promoter. Optimization of the sequence surrounding thetranslation initiation site of the reporter gene (GUS) resulted in afour-fold increase of GUS expression in transformed rice cells. Acombination of optimized translation initiation site and Act1 intronresulted in a 40-fold increase in GUS expression via the CaMV35Spromoter in transformed rice cells; similar results were achieved on thebasis of transformed corn cells. Overall, it was concluded from theabove-described studies that the expression vectors based on the Act1promoter are suitable for controlling a sufficiently strong andconstitutive expression of foreign DNA in transformed cells ofmonocotyledonous plants.

The expression cassette may advantageously contain one or more “enhancersequences” which are functionally linked to the promoter and whichenable an increased transgenic expression of the nucleic acid sequence.It is possible to insert additional advantageous sequences such asfurther regulatory elements or terminators at the 3′-end of the nucleicacid sequences to be expressed transgenically, too. Any of theexpression cassettes of the invention may contain one or more copies ofthe nucleic acid sequences to be expressed transgenically.

Control sequences furthermore means those which enable homologousrecombination or insertion into the genome of a host organism or whichallow the removal from the genome. In homologous recombination, forexample, the natural promoter of a particular gene may be replaced withone of the promoters of the invention. Methods such as the cre/loxtechnology allow tissue-specific, specifically inducible removal of theexpression cassette from the genome of the host organism (Sauer B.(1998) Methods. 14(4):381-92). In this case, particular flankingsequences are attached to the target gene (lox sequences), which laterenable a removal by means of the cre recombinase.

The promoter to be introduced may be placed upstream of the target geneto be expressed transgenically by means of homologous recombination bylinking the promoter to DNA sequences which are, for example, homologousto endogenous sequences upstream of the reading frame of the targetgene. Such sequences are to be understood as genetic control sequences.After a cell has been transformed with the appropriate DNA construct,the two homologous sequences can interact and thus place the promotersequence at the desired position upstream of the target gene so thatsaid promoter sequence is now functionally linked to the target gene andforms an expression cassette of the invention. The selection of thehomologous sequences determines the insertion point of the promoter. Inthis case, the expression cassette can be generated by homologousrecombination by means of a simple or a doubly-reciprocal recombination.In the case of the singly-reciprocal recombination, only a singlerecombination sequence is used and the entire introduced DNA isinserted. In the case of the doubly-reciprocal recombination, the DNA tobe introduced is flanked by two homologous sequences and the flankedregion is inserted. The latter method is suitable for replacing, asdescribed above, the natural promoter of a particular gene with one ofthe promoters of the invention and thus modifying the location and timeof expression of this gene. This functional linkage represents anexpression cassette of the invention.

The selection of successfully homologously recombined or elsetransformed cells normally requires the additional introduction of aselectable marker which imparts to the successfully recombined cells aresistance to a biocide (for example a herbicide), a metabolisminhibitor such as 2-desoxyglucose 6-phosphate (WO 98/45456) or to anantibiotic. The selection marker permits selection of the transformedcells from the untransformed cells (McCormick et al., Plant Cell Reports5 (1986), 81-84).

Homologous recombination is a relatively rare event in highereukaryotes, especially in plants. Random integrations into the hostgenome predominate. One possibility of removing the randomly integratedsequences and thus accumulating cell clones having a correct homologousrecombination is the use of a sequence-specific recombination system asdescribed in U.S. Pat. No. 6,110,736. This system consists of threeelements: two pairs of specific recombination sequences and asequence-specific recombinase. This recombinase catalyzes arecombination merely between the two pairs of specific recombinationsequences. One pair of these specific DNA sequences is placed outsidethe DNA sequence to integrated, i.e. outside the two homologous DNAsequences. In the case of a correct homologous recombination, thesesequences are not cotransferred into the genome. In the case of a randomintegration, they normally insert together with the rest of theconstruct. Using a specific recombinase and a construct comprising asecond pair of said specific sequences, the randomly inserted sequencescan be excised or inactivated by inversion, while the sequences insertedcorrectly via homologous recombination remain in the genome. It ispossible to use a multiplicity of sequence-specific recombinationsystems and the Cre/lox system of bacteriophage P1, the FLP/FRT systemof yeast, the Gin recombinase of phase Mu, the E. coli Pin recombinaseand the R/RS system of the plasmid pSR1 are mentioned by way of example.Preference is given to the bacteriophage P1 Cre/10.times. and the yeastFLP/FRT system. Here the recombinase (Cre or FLP) interacts specificallywith its respective recombination sequences (34 bp lox sequence or 47 bpFRT sequence) in order to delete or invert the transiently storedsequences. The FLP/FRT and cre/lox recombinase systems have already beenapplied to plant systems (Odell et al. (1990) Mol. Gen. Genet.,223:369-378).

Polyadenylation signals suitable as control sequences are plantpolyadenylation signals and, preferably, those which correspondessentially to Agrobacterium tumefaciens T-DNA polyadenylation signals,in particular of the T-DNA gene 3 (octopene synthase) of the Ti plasmidpTiACHS (Gielen et al., (1984) EMBO J. 3:(1984), 835 ff) or functionalequivalents thereof.

In a particularly preferred embodiment, the expression cassette containsa terminator sequence functional in plants. Terminator sequencesfunctional in plants means in general those sequences which are capableof causing the termination of transcription of a DNA sequence in plants.Examples of suitable terminator sequences are the OCS (octopenesynthase) terminator and the NOS (nopaline synthase) terminator.However, particular preference is given to terminator sequences ofplants. Terminator sequences of plants means in general those sequenceswhich are part of a natural plant gene. In this connection, particularpreference is given to the terminator of the potato cathepsin Dinhibitor gene (GenBank Acc. No.: X74985; terminator: SEQ ID NO: 28) orof the terminator of the field bean storage protein gene VfLEIB3(GenBank Ace. No.: Z26489; terminator: SEQ ID NO: 29). These terminatorsare at least equivalent to the viral or T-DNA terminators described inthe prior art. The plasmid pSUN5NPTIICat (SEQ ID NO: 24) contains theplant terminator of the potato cathepsin D inhibitor gene.

The skilled worker knows a multiplicity of nucleic acids or proteinswhose recombinant expression which is controlled by the expressioncassettes or methods of the invention is advantageous. The skilledworker further knows a multiplicity of genes whose repression orelimination by means of expression of a corresponding antisense RNA canlikewise achieve advantageous effects. Advantageous effects which may bementioned by way of example and not by way of limitation are:

-   -   easier preparation of a transgenic organism, for example by        expression of selection markers:    -   achieving a resistance to abiotic stress factors (heat, cold,        drought, increased humidity, environmental toxins, UV        radiation);    -   achieving a resistance to biotic stress factors (pathogens,        viruses, insects and diseases);    -   improvement of the properties of food- or feedstuffs;    -   improvement of growth rate or yield.

Some specific examples of nucleic acids whose expression provides thedesired advantageous effects are mentioned below:

1. Selection Markers.

Selection markers includes both positive selection markers which imparta resistance to an antibiotic, herbicide or biocide and negativeselection markers which impart a sensitivity to exactly these substancesand also markers which give a growth advantage to the transformedorganism (for example by expressing key genes of cytokine biosynthesis;Ebinuma H et al. (2000) Proc Natl Acad Sci USA 94:2117-2121). In thecase of positive selection, only those organisms which express theappropriate selection marker grow, while the same organisms die in thecase of negative selection. The preparation of transgenic plants prefersthe use of a positive selection marker. Furthermore, preference is givento using selection markers which impart growth advantages. Negativeselection markers may be used advantageously if particular genes orgenome sections are to be removed from an organism (for example in acrossing process).

The selectable marker introduced with the expression cassette imparts tothe successfully recombined or transformed cells a resistance to abiocide (for example a herbicide such as phosphinothricin, glyphosate orbromoxynil), a metabolism inhibitor such as 2-desoxyglucose 6-phosphate(WO 98/45456) or to an antibiotic such as, for example, kanamycin, G418, bleomycin, hygromycin. The selection marker permits selection ofthe transformed cells from the untransformed cells (McCormick et al.,Plant Cell Reports 5 (1986), 81-84). Particularly preferred selectionmarkers are those which impart a resistance to herbicides. A largenumber of such selection markers and the sequences coding therefor areknown to the skilled worker. Examples which may be mentioned by way ofexample but not by way of limitation are the following:

i) Positive Selection Markers.

The selectable marker introduced with the expression cassette imparts tothe successfully recombined or transformed cells a resistance to abiocide (for example a herbicide such as phosphinothricin, glyphosate orbromoxynil), a metabolism inhibitor such as 2-desoxyglucose 6-phosphate(WO 98/45456) or to an antibiotic such as, for example, tetracyclines,ampicillin, kanamycin, G418, neomycin, bleomycin or hygromycin. Theselection marker permits selection of the transformed cells from theuntransformed cells (McCormick et al., Plant Cell Reports 5 (1986),81-84). Particularly preferred selection markers are those which imparta resistance to herbicides. Examples of selection markers which may bementioned are:

-   -   DNA sequences coding for phosphinothricin acetyltransferases        (PAT) which acetylate the free amino group of the glutamine        synthase inhibitor phosphinothricin (PPT) and thus detoxify PPT        (de Block et al. 1987, EMBO J. 6, 2513-2518) (also referred to        as Bialaphos®-Resistance gene (bar)). The bar gene coding for a        phosphinothricin acetyltransferase (PAT) may be isolated, for        example, from Streptomyces hygroscopicus or S.        viridochromogenes. Corresponding sequences are known to the        skilled worker (from Streptomyces hygroscopicus GenBank Acc.        No.: X17220 and X05822, from Streptomyces viridochromogenes        GenBank Acc. No.: M 22827 and X65195; U.S. Pat. No. 5,489,520).        Furthermore, synthetic genes, for example for expression in        plastids, have been described AJ028212. A synthetic Pat gene is        described in Becker et al. (1994), The Plant J. 5:299-307. Very        particular preference is likewise given to the expression of the        polypeptide according to SEQ ID NO: 5, for example encoded by a        nucleic acid sequence according to SEQ ID NO: 4. The genes        impart a resistance to the herbicide Bialaphos® or glufosinate        and are frequently used markers in transgenic plants (Vickers, J        E et al. (1996). Plant Mol. Biol. Reporter 14:363-368; Thompson        C J et al. (1987) EMBO Journal 6:2519-2523).    -   5-Enolpyruvylshikimate 3-phosphate synthase genes (EPSP        synthasegenes) which impart a resistance to Glyphosat®        (N-(phosphonomethyl)glycin). The molecular target of the        unselective herbicide glyphosate is        5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS). This enzyme        has a key function in the biosynthesis of aromatic amino acids        in microbes and plants but not in mammals (Steinrucken H C et        al. (1980) Biochem. Biophys. Res. Commun. 94:1207-1212; Levin J        G and Sprinson D B (1964) J. Biol. Chem. 239: 1142-1150; Cole D        J (1985) Mode of action of glyphosate; A literature analysis, p.        48-74. In: Grossbard E and Atkinson D (eds.). The herbicide        glyphosate. Buttersworths, Boston). Preference is given to using        glyphosate-tolerant EPSPS variants as selection markers        (Padgette S R et al. (1996). New weed control opportunities:        development of soybeans with a Roundup Ready™ gene. In:        Herbicide Resistant Crops (Duke, S. O., ed.), pp. 53-84. CRC        Press, Boca Raton, Fla.; Saroha M K and Malik V S (1998) J Plant        Biochemistry and Biotechnology 7:65-72). The EPSPS gene of        Agrobacterium sp. strain CP4 has a natural tolerance for        glyphosate, which can be transferred to appropriate transgenic        plants. The CP4 EPSPS gene was cloned from Agrobacterium sp.        strain CP4 (Padgette S R et al. (1995) Crop Science        35(5):1451-1461). 5-Enolpyruvylshikimate 3-phosphate synthases,        which are glyphosate-tolerant, as described, for example, in        U.S. Pat. No. 5,510,471; U.S. Pat. No. 5,776,760; U.S. Pat. No.        5,864,425; U.S. Pat. No. 5,633,435; U.S. Pat. No. 5,627,061;        U.S. Pat. No. 5,463,175; EP 0 218 571, are preferred and the        sequences described in each case in the patents have also been        deposited with GenBank. Further sequences are described under        GenBank Accession X63374. The aroA gene is also preferred        (M10947 S. typhimurium aroA locus        5-enolpyruvylshikimate-3-phosphate synthase (aroA protein)        gene).    -   the gox (glyphosate oxidoreductase) gene coding for the        Glyphosat®-degrading enzyme. GOX (for example Achromobacter sp.        glyphosate oxidoreductase) catalyzes the cleavage of a C—N bond        in glyphosate which is thus converted to aminomethylphosphonic        acid (AMPA) and glyoxylate. GOX can thereby mediate a resistance        to glyphosate (Padgette S R et al. (1996) J. Nutr. 1996 March;        126(3):702-16; Shah D et al. (1986) Science 233: 478-481).    -   the deh gene (coding for a dehalogenase which inactivates        Dalaponr), (GenBank Acc. No.: AX022822, AX022820 and        WO99/27116).    -   bxn genes which code for Bromoxynil®-degrading nitrilase enzyme.        For example the Klebsiella ozanenae nitrilase. Sequences can be        found at GenBank, for example under Acc. No: EO 1313 (DNA        encoding bromoxynil-specific nitrilase) and J03196 (K.        pneumoniae bromoxynil-specific nitrilase (bxn) gene, complete        cds).    -   Neomycin phosphotransferases impart a resistance to antibiotics        (aminoglycosides) such as neomycin, G418, hygromycin,        paromomycin or kanamycin by reducing the inhibiting action        thereof by a phosphorylation reaction. Particular preference is        given to the nptII gene. Sequences can be obtained from GenBank        (AF080390 minitransposon mTn5-GNm; AF080389 minitransposon        mTn5-Nm, complete sequence). Moreover, the gene is already part        of numerous expression vectors and can be isolated therefrom by        using methods familiar to the skilled worker (such as, for        example, polymerase chain reaction) (AF234316 pCAMBIA-2301;        AF234315 pCAMBIA-2300, AF234314 pCAMBIA-2201). The NPTII gene        codes for an aminoglycoside 3′-O-phosphotransferase from E.        coli, Tn5 (GenBank Acc. No: U00004 position 1401-2300; Beck et        al. (1982) Gene 19 327-336).    -   the DOG®1-gene. The DOG®1 gene was isolated from the yeast        Saccharomyces cerevisiae (EP 0 807 836). It codes for a        2-desoxyglucose 6-phosphate phosphatase which imparts resistance        to 2-DOG (Randez-Gil et al. 1995, Yeast 11, 1233-1240; Sanz et        al. (1994) Yeast 10:1195-1202, Sequence: GenBank Ace. No.:        NC001140 chromosome VIII, Saccharomyces cervisiae position        194799-194056).    -   Sulfonylurea- and imidazolinone-inactivating acetolactate        synthases which impart a resistance to        imidazolinone/sulfonylurea herbicides. Examples of imidazolinone        herbicides which may be mentioned are the active substances        imazamethabenz-methyl, imazamox, imazapyr, imazaquin,        imazethapyr. Examples of sulfonylurea herbicides which may be        mentioned are amidosulfuron, azimsulfuron, chlorimuronethyl,        chlorsulfuron, cinosulfuron, imazosulfuron, oxasulfuron,        prosulfuron, rimsulfuron, sulfosulfuron. Numerous further active        substances of said classes are known to the skilled worker. An        example of a suitable sequence is the sequence of Arabidopsis        thaliana Csr 1.2 gene deposited under the GenBank Acc-No.:        X51514 (EC 4.1.3.18) (Sathasivan K et al. (1990) Nucleic Acids        Res. 18(8):2188). Acetolactate synthases which impart a        resistance to imidazolinon herbicides are furthermore described        under GenBank Ace. Nos.:

a) AB049823 Oryza sativa ALS mRNA for acetolactate synthase, completecds, herbicide resistant biotype;

b) AF094326 Bassia scoparia herbicide resistant acetolactate synthaseprecursor (ALS) gene, complete cds;

c) X07645 Tobacco acetolactate synthase gene, ALS SuRB (EC 4.1.3.18);

d) X07644 Tobacco acetolactate synthase gene, ALS SuRA (EC 4.1.3.18);

e) A19547 Synthetic nucleotide mutant acetolactate synthase;

f) A19546 Synthetic nucleotide mutant acetolactate synthase;

g) A19545 Synthetic nucleotide mutant acetolactate synthase;

h) 105376 Sequence 5 from Patent EP 0257993;

i) Sequence 2 from Patent EP 0257993;

j) AL133315.

Preference is given to expressing an acetolactate synthase according toSEQ ID NO: 7, for example encoded by a nucleic acid sequence accordingto SEQ ID NO: 6.

-   -   Hygromycin phosphotransferases (X74325 P. pseudomallei gene for        hygromycin phosphotransferase) which impart a resistance to the        antibiotic hygromycin. The gene is part of numerous expression        vectors and can be isolated therefrom by using methods familiar        to the skilled worker (such as, for example, polymerase chain        reaction) (AF294981 pINDEX4; AF234301 pCAMBIA-1380; AF234300        pCAMBIA-1304; AF234299 pCAMBIA-1303; AF234298 pCAMBIA-1302;        AF354046 pCAMBIA-1305; AF354045 pCAMBIA-1305.1).    -   Genes for resistance to:

a) chloramphenicol (chloramphenicol acetyltransferase);

tetracycline, various resistance genes have been described, for exampleX65876 S. ordonez genes class D tetA and tetR for tetracyclineresistance and repressor proteins X51366 Bacillus cereus plasmid pBC16tetracycline resistance gene. The gene is also already part of numerousexpression vectors and can be isolated therefrom by using methodsfamiliar to the skilled worker (such as, for example, polymerase chainreaction);

c) streptomycin, various resistance genes have been described, forexample under GenBank Acc. No.: AJ278607 Corynebacteriumacetoacidophilum ant gene for streptomycin adenylyltransferase;

d) zeocin, the corresponding resistance gene is part of numerous cloningvectors (e.g. L36849 cloning vector pZEO) and can be isolated therefromby using methods familiar to the skilled worker (such as, for example,polymerase chain reaction);

e) ampicillin (β-lactamase gene; Datta N, Richmond M H. (1966) BiochemJ. 98(1):204-9; Heffron F et al (1975) J. Bacteriol 122: 250-256; theamp gene was initially cloned for preparing the E. coli vectors pBR322;Bolivar F et al. (1977) Gene 2:95-114). The sequence is part of numerouscloning vectors and can be isolated therefrom by using methods familiarto the skilled worker (such as, for example, polymerase chain reaction).

-   -   Genes such as the isopentenyl transferase from Agrobacterium        tumefaciens (strain:PO22) (GenBank Acc. No.: AB025109). The ipt        gene is [lacuna] a key enzyme of cytokine biosynthesis. Its        overexpression facilitates the regeneration of plants (e.g.        selection of cytokine-free medium). The method for using the ipt        gene has been described (Ebinuma H et al. (2000) Proc Natl Acad        Sci USA 94:2117-2121; Ebinuma, H et al. (2000) Selection of        Marker-free transgenic plants using the oncogenes (ipt, rol A,        B, C) of Agrobacterium as selectable markers, In Molecular        Biology of Woody Plants. Kluwer Academic Publishers).

Various other positive selection markers which impart to the transformedplants a growth advantage over untransformed plants and methods of theiruse are described, inter alia, in EP-A 0 601 092. Examples which may bementioned are β-glucuronidase (in connection with, for example,cytokinine glucuronide), mannose 6-phosphate isomerase (in connectionwith mannose), UDP-galactose 4-epimerase (in connection with, forexample, galactose), mannose 6-phosphate isomerase in connection withmannose being particularly preferred.

ii) Negative Selection Markers.

Negative selection markers make possible, for example, the selection oforganisms in which sequences comprising the marker gene have beensuccessfully deleted (Koprek T et al. (1999) The Plant Journal19(6):719-726). In negative selection, for example, a compound whichotherwise has no disadvantageous effect on the plant is converted to acompound having a disadvantageous effect, due to the negative selectionmarker introduced into the plant. Genes which have a disadvantageouseffect per se, such as, for example, TK thymidine kinase (TK), anddiphtheria toxin A fragment (DT-A), the codA gene product coding for acytosine deaminase (Gleave A P et al. (1999) Plant Mol. Biol.40(2):223-35; Perera R J et al. (1993) Plant Mol. Biol. 23(4): 793-799;Stougaard J; (1993) Plant J 3:755-761), the cytochrom P450 gene (Kopreket al. (1999) Plant J. 16:719-726), genes coding for a haloalkanedehalogenase (Naested H (1999) Plant J. 18:571-576), the iaaH gene(Sundaresan V et al. (1995) Genes & Development 9:1797-1810) and thetms2 gene (Fedoroff N V & Smith D L 1993, Plant J 3: 273-289) are alsosuitable.

The concentrations of the antibiotics, herbicides, biocides or toxins,used in each case for selection, have to be adapted to the particularassay conditions or organisms. Examples which may be mentioned forplants are kanamycin (Km) 50 mg/l, hygromycin B 40 mg/l,phosphinothricin (Ppt) 6 mg/l.

It is furthermore possible to express functional analogs of said nucleicacids coding for selection markers. Functional analogs here means allthose sequences which have essentially the same function, i.e. which arecapable of selection of transformed organisms. In this connection, thefunctional analog may quite possibly differ in other features. It mayhave, for example, a higher or lower activity or else furtherfunctionalities.

2. Improved protection of the plant against abiotic stress factors suchas drought, heat or cold, for example by overexpression ofantifreeze-polypeptides from Myoxocephalus Scorpius (WO 00/00512),Myoxocephalus octodecemspinosus, of Arabidopsis thaliana transcriptionactivator CBF1, of glutamate dehydrogenases (WO 97/12983, WO 98/11240),calcium-dependent protein kinase genes (WO 98/26045), calcineurins (WO99/05902), farnesyl transferases (WO 99/06580, Pei Z M et al., Science1998, 282: 287-290), ferritin (Deak M et al., Nature Biotechnology 1999,17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M Biotechnology andGenetic Engineering Reviews 1998, 15:1-32), DREB1A-Factor (dehydrationresponse element B 1A; Kasuga M et al., Nature Biotechnology 1999,17:276-286), of genes of mannitol or trehalose synthesis, such astrehalose phosphate synthase or trehalose phosphate phosphatase (WO97/42326), or by inhibition of genes such as trehalose (WO 98/50561).Particular preference is given to nucleic acids which code for theArabidopsis thaliana transcription activator CBF1 (GenBank Acc. No.:U77378) or for the Myoxocephalus octodecemspinosus antifreeze protein(GenBank Acc. No.: AF306348) or functional equivalents of the same.

3. Expression of metabolic enzymes for use in the feed and food sectors,for example expression of phytase and cellulases. Particular preferenceis given to nucleic acids such as the artificial cDNA coding for amicrobial phytase (GenBank Acc. No.: A119451) or functional equivalentsthereof.

4. Achieving a resistance, for example to fungi, insects, nematodes anddiseases, by specific isolation or accumulation of particularmetabolites or proteins in the embryonic epidermis. Examples which maybe mentioned are glucosinolates (repulsion of herbivores), chitinases orglucanases and other enzymes which destroy the cell wall of parasites,ribosome-inactivating proteins (RIPs) and other proteins of resistanceand stress reactions of the plant, such as those induced by injury ormicrobial infection of plants or chemically by, for example, salicylicacid, jasmonic acid or ethylene, lysozymes from sources other thanplants, such as, for example, T4 lysozyme or lysozyme from variousmammals, insecticidal proteins such as Bacillus thuringiensis endotoxin,α-amylase inhibitor or protease inhibitors (cowpea trypsine inhibitor),glucanases, lectins such as phytohemagglutinin, snowdrop lectin, wheatgerm agglutinine, RNases and ribozymes. Particular preference is givento nucleic acids coding for chit42 endochitinase from Trichodermaharzianum (GenBank Acc. No.: S78423) or for the N-hydroxylating,multifunctional cytochrome P-450 (CYP79) protein from Sorghum bicolor(GenBank Am. No.: U32624) or functional equivalents thereof.

What is known is the accumulation of glucosinolates in plants of thegenus of Cardales, in particular of oilseeds, for protection againstpests (Rask L et al. (2000) Plant Mol Biol 42:93-113; Menard R et al.(1999) Phytochemistry 52:29-35), the expression of the Bacillusthuringiensis endotoxin under the control of the 35 S CaMV promoter(Vaeck et al. (1987) Nature 328:33-37) or the protection of tobaccoagainst fungal infection by expression of a bean chitinase under thecontrol of the CaMV promoter (Broglie et al. (1991) Science254:1194-1197).

The expression of the snowdrop (Galanthus nivalis) lectin agglutininecan achieve a resistance to pests such as the rice pest Nilaparvatalugens, for example in transgenic rice plants (Rao et al. (1998) PlantJ. 15(4):469-77.). Nilaparvata lugens belongs to the phloem-suckingpests and, in addition, acts as a transmitter of important virus-basedplant diseases.

The expression of synthetic cryIA(b) and cryIA(c) genes which code forlepidoptera-specific delta-entotoxins from Bacillus thuringiensis, cancause a resistance to insect pests in various plants. Thus it ispossible to achieve a resistance in rice to two of the most importantrice insect pests, the striped stem borer (Chilo suppressalis) and theyellow stem borer (Scirpophaga incertulas), (Cheng X et al. (1998) ProcNatl Acad Sci USA 95(6):2767-2772; Nayak P et al. (1997) Proc Natl AcadSci USA 94(6):2111-2116).

5. Expression of genes which cause accumulation of fine chemicals suchas tocopherols, tocotrienols or carotenoids. Phytoene desaturase may bementioned as an example. Preference is given to nucleic acids which codefor Narcissus pseudonarcissus phytoene desaturase (GenBank Acc. No.:X78815) or functional equivalents thereof.

6. Production of nutraceuticals such as, for example, polyunsaturatedfatty acids such as, for example, arachidonic acid or EP(eicosapentenoic acid) or DHA (docosahexaenoic acid) by expressingfatty-acid elongases and/or desaturases or by producing proteins havingan improved nutritional value such as, for example, a high proportion ofessential amino acids (e.g. the methionine-rich brazil nut albumingen).Preference is given to nucleic acids coding for the methionine-richBertholletia excelsa 2S albumin (GenBank Acc. No.: AB044391), thePhyscomitrella patens Δ6-acyllipid desaturase (GenBank Acc. No.:AJ222980; Girke et al 1998, The Plant Journal 15:39-48), the Mortierellaalpina Δ6-desaturase (Sakuradani et al 1999 Gene 238:445-453), theCaenorhabditis elegans Δ5-desaturase (Michaelson et al. 1998, FEBSLetters 439:215-218), the Caenorhabditis elegans Δ5-fatty-aciddesaturase (des-5) (GenBank Acc. No.: AF078796), the Mortierella alpinaΔ5-desaturase (Michaelson et al. JBC 273:19055-19059), theCaenorhabditis elegans Δ6-elongase (Beaudoin et al. 2000, PNAS97:6421-6426), the Physcomitrella patens Δ6-elongase (Zank et al. 2000,Biochemical Society Transactions 28:654-657) or functional equivalentsthereof.

7. Production of fine chemicals (such as, for example, enzymes) andpharmaceuticals (such as, for example, antibodies or vaccines, asdescribed in Hood E E, Jilka J M. (1999) Curr Opin Biotechnol.10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol236:275-92). For example, it was possible to produce on a large scalerecombinant avidin from egg white and bacterial .beta.-glucuronidase(GUS) in transgenic corn plants (Hood et al. (1999) Adv Exp Med Biol464:127-47. Review). These recombinant proteins from corn plants aresold by Sigma (Sigma Chemicals Co.) as high-purity biochemicals.

Achieving an increased storage capability in cells which usually containrelatively few storage proteins or storage lipids, with the aim ofincreasing the yield of said substances, for example by expressing anacetyl-CoA carboxylase. Preference is given to nucleic acids coding forMedicago sativa acetyl-CoA carboxylase (accase) (GenBank Acc. No.:L25042) or functional equivalents thereof

Further examples of advantageous genes are mentioned, for example, inDunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96. It is furthermore possible to expressfunctional analogs of the nucleic acids and proteins mentioned.Functional analogs here means all those sequences which have essentiallythe same function, i.e. which are capable of the same function (forexample substrate conversion or signal transduction) as the proteinmentioned by way of example. The functional analog may quite possiblydiffer in other features. It may have, for example, a higher or loweractivity or else have further functionalities. Functional analogsfurther means sequences which code for fusion proteins comprising one ofthe preferred proteins and other proteins, for example another preferredprotein, or else a signal peptide sequence.

The nucleic acids may be expressed under the control of the promoters ofthe invention in any desired cell compartment such as, for example, theendomembrane system, the vacuole and the chloroplasts. Desiredglycosylation reactions, particular foldings, and the like are possibleby utilizing the secretory pathway. Secretion of the target protein tothe cell surface or secretion into the culture medium, for example whenusing suspension-cultured cells or protoplasts, is also possible. Therequired target sequences may both be taken into account in individualvector variations and be introduced into the vector together with thetarget gene to be cloned by using a suitable cloning strategy. Targetsequences which may be used are both endogenous, if present, andheterologous sequences. Additional heterologous sequences which arepreferred for functional linkage but not limited thereto are furthertargeting sequences for ensuring subcellular localization in theapoplast, in the vacuole, in plastids, in mitochondria, in theendoplasmic reticulum (ER), in the nucleus, in elaioplasts or othercompartments; and also translation enhancers such as the 5′-leadersequence from tobacco mosaic virus (Gallie et al., Nucl. Acids Res. 15(1987), 8693-8711) and the like. The method of transporting proteinswhich are per se not located in the plastids specifically into saidplastids has been described, (Klosgen R B and Weil J H (1991) Mol GenGenet. 225(2):297-304; Van Breusegem F et al. (1998) Plant Mol. Biol.38(3):491-496).

Preferred Sequences are:

a) small subunit (SSU) of ribulose bisphosphate carboxylase (Rubiscossu) from pea, corn, sunflower;

b) transit peptides derived from genes of fatty-acid biosynthesis inplants, such as the transit peptide of the plastid acyl carrier protein(ACP), stearyl-ACP desaturase, β-ketoacyl-ACP synthase or acyl-ACPthioesterase;

c) the transit peptide for GBSSI (“granule bound starch synthase I”);and

d) LHCP II genes.

The target sequences may be linked to other targeting sequences whichdiffer from the transit peptide-encoding sequences, in order to ensuresubcellular localization in the apoplast, in the vacuole, in plastids,in the mitochondrion, in the endoplastic reticulum (ER), in the nucleus,in elaioplasts or in other compartments. It is also possible to usetranslation enhancers such as the 5′-leader sequence from tobacco mosaicvirus (Gallie et al. (1987), Nucl. Acids Res. 15: 8693-8711) and thelike.

The skilled worker further knows that there is no need for him toexpress the above-described genes directly by using the nucleic acidsequences coding for said genes or to repress them by antisense, forexample. He may also use, for example, artificial transcription factorsof the zinc finger protein type (Beerli R R et al. (2000) Proc Natl AcadSci USA 97(4):1495-500). These factors attach to the regulatory regionsof the endogenous genes to be expressed or repressed and causeexpression or repression of the endogenous gene, depending on the designof the factor. Thus it is also possible to achieve the desired effectsby expressing an appropriate zinc finger transcription factor under thecontrol of one of the promoters of the invention.

It is likewise possible to use the expression cassettes of the inventionfor suppressing or reducing the replication or/and translation of targetgenes by gene silencing.

The expression cassettes of the invention may also be employed forexpressing nucleic acids which mediate “antisense” effects and thus arecapable of reducing the expression of a target protein, for example.

Preferred genes and proteins whose suppression results in anadvantageous phenotype include by way of example but not by way oflimitation:

a) polygalacturonase for preventing cell degradation and preventingplants and fruits, for example tomatoes, from becoming “mushy”.Preference is given to using for this nucleic acid sequences such asthat of the tomato polygalacturonase gene (GenBank Acc. No.: X14074) orits homologs from other genera and species.

reducing the expression of allergenic proteins, as described, forexample, in Tada Y et al. (1996) FEBS Lett 391(3):341-345 or Nakamura R(1996) Biosci Biotechnol Biochem 60(8):1215-1221.

c) modifying the color of flowers by suppressing the expression ofenzymes of anthocyane biosynthesis. Appropriate procedures have beendescribed (for example in Forkmann G, Martens S. (2001) Curr OpinBiotechnol 12(2):155-160). Preference is given to using for this nucleicacid sequences such as those of flavonoid 3′-hydroxylase (GenBank Acc.No.: AB045593), dihydroflavanol 4-reductase (GenBank Acc. No.:AF017451), chalcone isomerase (GenBank Acc. No.: AF276302), chalconesynthase (GenBank Acc. No.: AB061022), flavanone 3-beta-hydroxylase(GenBank Acc. No.: X72592) and flavone synthase II (GenBank Acc. No.:AB045592) and the homologs thereof from other genera and species.

d) altering the amylose/amylopectin content in starch by suppressing thebranching enzyme Q which is responsible for the α-1,6-glycosidiclinkage. Appropriate procedures have been described (for example inSchwall G P et al. (2000) Nat Biotechnol 18(5):551-554). Preference isgiven to using for this nucleic acid sequences such as that of thepotato starch branching enzyme II (GenBank Ace. No.: AR123356; U.S. Pat.No. 6,169,226) or its homologs from other genera and species.

An antisense nucleic acid first means a nucleic acid sequence which iscompletely or partially complementary to at least a part of the sensestrand of said target protein. The skilled worker knows that it ispossible to use, as an alternative, the cDNA or the corresponding geneas starting template for corresponding antisense constructs. Preferably,the antisense nucleic acid is complementary to the coding region of thetarget protein or to a part thereof. However, the antisense nucleic acidmay also be complementary to the noncoding region or to a part thereof.Starting from the sequence information for a target protein, it ispossible to design an antisense nucleic acid in the manner familiar tothe skilled worker by taking into account the Watson and Crick basepairing rules. An antisense nucleic acid may be complementary to theentire or to a part of the nucleic acid sequence of a target protein. Ina preferred embodiment, the antisense nucleic acid is an oligonucleotideof, for example, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

In a preferred embodiment, the antisense nucleic acid comprisesα-anomeric nucleic acid molecules. α-anomeric nucleic acid moleculesform particular double-stranded hybrids with complementary RNA, inwhich, in contrast to the normal β-units, the strands run parallel toone another (Gautier et al. (1987) Nucleic Acids. Res. 15:6625-6641).

Likewise included is the use of the above-described sequences in senseorientation, which may lead to cosuppression, as is familiar to theskilled worker. It has been demonstrated in tobacco, tomato and petuniathat expression of sense RNA of an endogenous gene can reduce oreliminate expression of said gene, in a similar manner to what has beendescribed for antisense approaches (Goring et al. (1991) Proc. Natl.Acad Sci USA, 88:1770-1774; Smith et al. (1990) Mol Gen Genet.224:447-481; Napoli et al. (1990) Plant Cell 2:279-289; Van der Krol etal. (1990) Plant Cell 2:291-299). The introduced construct may representthe gene to be reduced completely or only partially. The possibility oftranslation is not required.

Very particular preference is also given to the use of methods such asgene regulation by means of double-stranded RNA (double-stranded RNAinterference). Relevant methods are known to the skilled worker and havebeen described in detail (e.g. Matzke M A et al. (2000) Plant Mol Biol43:401-415; Fire A. et al (1998) Nature 391:806-811; WO 99/32619; WO99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO00/63364). The processes and methods described in the references listedare hereby expressly incorporated by reference. The simultaneousintroduction of strand and complementary strand causes here a highlyefficient suppression of native genes.

Advantageously, the antisense strategy may be coupled with a ribozymemethod. Ribozymes are catalytically active RNA sequences which, coupledto the antisense sequences, catalytically cleave the target sequences(Tanner N K. FEMS Microbiol Rev. 1999; 23 (3):257-75). This can increasethe efficiency of an antisense strategy. The expression of ribozymes inorder to reduce particular proteins is known to the skilled worker andis described, for example, in EP-A1 0 291 533, EP-A1 0 321 201 and EP-A10 360 257. Suitable target sequences and ribozymes may be determined,for example, as described in Steinecke (Ribozymes, Methods in CellBiology 50, Galbraith et al eds Academic Press, Inc. (1995), 449-460),by calculations of the secondary structure of ribozyme RNA and targetRNA and by the interaction thereof (Bayley C C et al., Plant Mol. Biol.1992; 18(2):353-361; Lloyd A M and Davis R W et al., Mol Gen Genet. 1994March; 242(6):653-657). An example which may be mentioned is hammerheadribozymes (Haselhoff and Gerlach (1988) Nature 334:585-591). Preferredribozymes are based on derivatives of Tetrahymena L-19 IVS RNA (U.S.Pat. No. 4,987,071; U.S. Pat. No. 5,116,742). Further ribozymes withselectivity for an L119 mRNA may be selected (Bartel D and Szostak J W(1993) Science 261:1411-1418).

In another embodiment, target protein expression may be reduced usingnucleic acid sequences which are complementary to regulatory elements ofthe target protein genes and which form together with said genes atriple-helical structure and thus prevent gene transcription (Helene C(1991) Anticancer Drug Des. 6(6):569-84; Helene C et al. (1992) Ann NYAcad Sci 660:27-36; Maher L J (1992) Bioassays 14(12):807-815).

The expression cassette of the invention and the vectors derivedtherefrom may contain further functional elements.

The term functional element has a broad meaning and means all thoseelements which influence preparation, propagation or function of theexpression cassettes of the invention or of vectors or organisms derivedtherefrom. Examples which may be mentioned but which are not limitingare:

reporter genes which code for readily quantifiable proteins and whichensure, via intrinsic color or enzyme activity, an evaluation of thetransformation efficiency and of the location or time of expression. Inthis connection, very particular preference is given to genes coding forreporter proteins (see also Schenborn E, Groskreutz D. Mol. Biotechnol.1999; 13(1):29-44) such as:

green fluorescence protein (GFP) (Chui W L et al., Curr Biol 1996,6:325-330; Leffel S M et al., Biotechniques. 23(5):912-8, 1997; Sheen etal. (1995) Plant Journal 8(5):777-784; Haseloff et al. (1997) Proc NatlAcad Sci USA 94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad SciUSA 93(12):5888-5893; Tian et al. (1997) Plant Cell Rep 16:267-271; WO97/41228);

chloramphenicol transferase (Fromm et al. (1985) Proc. Natl. Acad. Sci.USA 82:5824-5828);

Luciferase (Millar et al., Plant Mol Biol Rep 1992 10:324-414; Ow et al.(1986) Science, 234:856-859); allows bioluminescence detection;

β-galactosidase, coding for an enzyme for which various chromogenicsubstrates are available;

β-glucuronidase (GUS) (Jefferson et al., EMBO J. 1987, 6, 3901-3907) orthe uidA gene which encodes an enzyme for various chromogenicsubstrates;

R-locus gene product: protein which regulates production of anthocyaninepigments (red color) in plant tissue and thus makes possible a directanalysis of the promoter activity without the addition of additionalauxiliary substances or chromogenic substrates (Dellaporta et al., In:Chromosome Structure and Function: Impact of New Concepts, 18th StadlerGenetics Symposium, 11:263-282, 1988);

β-lactamase (Sutcliffe (1978) Proc Natl Acad Sci USA 75:3737-3741),enzyme for various chromogenic substrates (e.g. PADAC, a chromogeniccephalosporin);

xylE gene product (Zukowsky et al. (1983) Proc Natl Acad Sci USA80:1101-1105), catechol dioxygenase which can convert chromogeniccatechols;

alpha-amylase (Ikuta et al. (1990) Bio/technol. 8:241-242);

tyrosinase (Katz et al. (1983) J Gen Microbiol 129:2703-2714), enzymewhich oxidizes tyrosine to give DOPA and dopaquinone which consequentlyform the readily detectable melanine;

aequorin (Prasher et al. (1985) Biochem Biophys Res Commun126(3):1259-1268), may be used in calcium-sensitive bioluminescencedetection.

replication origins which ensure a propagation of the expressioncassettes or vectors of the invention, for example in E. coli. Exampleswhich may be mentioned are ORI (origin of DNA replication), the pBR322ori or the P15A ori (Sambrook et al.: Molecular Cloning. A LaboratoryManual, 2^(nd) ed. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

elements, for example border sequences, which enableagrobacteria-mediated transfer into plant cells for transfer andintegration into the plant genome, such as, for example, the right orleft border of T-DNA or the vir region.

multiple cloning regions (MCS) allow and facilitate the insertion of oneor more nucleic acid sequences.

Various ways to achieve an expression cassette of the invention areknown to the skilled worker. An expression cassette of the invention isprepared, for example, by fusing one of the promoters of the invention(or a functional equivalent or functionally equivalent part according toSEQ ID NO: 1, 2 or 3) or a functional equivalent to a nucleotidesequence to be expressed, where appropriate to a sequence coding for atransit peptide, preferably a chloroplast-specific transit peptide,which is preferably located between the promoter and the particularnucleotide sequence, and also with a terminator or polyadenylationsignal. For this purpose, common recombination and cloning techniques asdescribed, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman andL. W. Enquist, Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. etal., (1987) Current Protocols in Molecular Biology, Greene PublishingAssoc. and Wiley Interscience are used.

However, an expression cassette means also those constructs in which thepromoter without having been functionally linked beforehand to a nucleicacid sequence to be expressed, is introduced into a host genome, forexample, via specific homologous recombination or random insertion andtakes over there regulatory control over nucleic acid sequences thenfunctionally linked to it and controls transgenic expression of saidnucleic acid sequences. Insertion of the promoter, for example byhomologous recombination, upstream of a nucleic acid coding for aparticular polypeptide results in an expression cassette of theinvention, which controls expression of the particular polypeptide inthe plant. Furthermore, the promoter may also be inserted such thatantisense RNA of the nucleic acid coding for a particular polypeptide isexpressed. As a result, the expression of said particular polypeptide inplants is down-regulated or eliminated.

Analogously, it is also possible to place a nucleic acid sequence to beexpressed transgenically downstream of the endogenous natural promoter,for example by homologous recombination, resulting in an expressioncassette of the invention, which controls expression of the nucleic acidsequence to be expressed transgenically in the cotyledons of the plantembryo.

The invention further relates to vectors which contain theabove-described expression cassettes. Vectors may be, by way of example,plasmids, cosmids, phages, viruses or else agrobacteria.

The invention also relates to transgenic organisms transformed with atleast one expression cassette of the invention or one vector of theinvention and also to cells, cell cultures, tissue, parts, such as, forexample in the case of plant organisms, leaves, roots, etc., orpropagation material derived from such organisms.

Organisms, starting or host organisms mean prokaryotic or eukaryoticorganisms such as, for example, microorganisms or plant organisms.Preferred microorganisms are bacteria, yeasts, algae or fungi. Preferredbacteria are bacteria of the genus Escherichia, Erwinia, Agrobacterium,Flavobacterium, Alcaligenes or cyanobacteria for example of the genusSynechocystis. Preference is given especially to microorganisms whichare capable of infecting plants and thus transferring the cassettes ofthe invention. Preferred microorganisms are those of the genusAgrobacterium and, in particular of the species Agrobacteriumtumefaciens. Preferred yeasts are Candida, Saccharomyces, Hansenula andPichia. Preferred fungi are Aspergillus, Trichoderma, Ashbya,Neurospora, Fusarium, Beauveria or other fungi described in Indian Chem.Engr. Section B. Vol 37, No 1,2 (1995) on page 15, Table 6.

Host or starting organisms preferred as transgenic organisms areespecially plants. Included within the scope of the invention are allgenera and species of the higher and lower plants of the plant kingdom.The mature plants, seeds, shoots and seedlings and also parts,propagation material and cultures, for example cell cultures, derivedtherefrom are also included. Mature plants means plants at anydevelopment stage beyond the seedling. Seedling means a young immatureplant in an early development stage.

Annual, perennial, monocotyledonous and dicotyledonous plants arepreferred host organisms for preparing transgenic plants. The expressionof genes is furthermore advantageous in all ornamental plants, useful orornamental trees, flowers, cut flowers, shrubs or lawns. Plants whichmay be mentioned by way of example but not by limitation areangiosperms, bryophytes such as, for example, Hepaticae (liverworts) andMusci (mosses); pteridophytes such as ferns, horsetail and club mosses;gymnosperms such as conifers, cycades, ginkgo and Gnetalae; algae suchas Chlorophyceae, Phaeophpyceae, Rhodophyceae, Myxophyceae,Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae.

Preference is given to plants of the following plant families:Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae,Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae,Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae,Saxifragaceae, Scrophulariaceae, Solanacea, Sterculiaceae,Tetragoniacea, Theaceae, Umbelliferae. Preferred monocotyledonous plantsare in particular selected from the monocotyledonous crop plants, forexample of the Gramineae family, such as rice, corn, wheat, or othercereal species such as barley, malt, rye, triticale or oats, and alsosugar cane and all grass species. Preferred dicotyledonous plants are inparticular selected from the dicotyledonous crop plants, for example:Asteraceae such as sunflower, Tagetes or Calendula and others,Compositae, particularly the genus Lactuca, in particular the speciessativa (lettuce), and others, Cruciferae, particularly the genusBrassica, very particularly the species napus (oilseed rape), campestris(beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y(cauliflower) and oleracea cv Emperor (broccoli), and further cabbagespecies; and the genus Arabidopsis, very particularly the speciesthaliana, and also cress or canola, and others, Cucurbitaceae such asmelon, pumpkin or zucchini, and others, Leguminosae particularly thegenus Glycine, very particularly the species max (soyabean), soya andalso alfalfa, pea, bean plants or peanut, and others, Rubiaceae,preferably of the subclass Lamidae, such as, for example, Coffea arabicaor Coffea liberica (coffee bush), and others, Solanaceae, in particularthe genus Lycopersicon, very particularly the species esculentum(tomato), and the genus Solanum, very particularly the species tuberosum(potato) and melongena (aubergine) and also tobacco or paprika, andothers, Sterculiaceae, preferably of the subclass Dilleniidae, such as,for example, Theobroma cacao (cacao bush) and others, Theaceae,preferably of the subclass Dilleniidae, such as, for example, Camelliasinensis or Thea sinensis (tea shrub) and others, Umbelliferae,preferably the genus Daucus, very particularly the species carota(carrot), and Apium (very particularly the species graveolens dulce(celery), and others; and the genus Capsicum, very particularly thespecies annum (pepper), and others, and also linseed, soya, cotton,hemp, flax, cucumber, spinach, carrot, sugarbeet and the various tree,nut and vine species, in particular banana and kiwi fruit.

Also included are ornamental plants, useful and ornamental trees,flowers, cut flowers, shrubs and lawns. Plants which may be mentioned byway of example but not by limitation are angiosperms, bryophytes suchas, for example, Hepaticae (liverworts) and Musci (mosses);pteridophytes such as ferns, horsetail and club mosses; gymnosperms suchas conifers, cycades, ginkgo and Gnetalae, the Rosaceae families, suchas rose, Ericaceae such as rhododendrons and azaleas, Euphorbiaceae suchas poinsettias and croton, Caryophyllaceae such as pinks, Solanaceaesuch as petunias, Gesneriaceae such as African violet, Balsaminaceaesuch as catch-me-not, Orchidaceae such as orchids, Iridaceae such asgladioli, iris, freesia and crocus, Compositae such as marigold,Geraniaceae such as gerania, Liliaceae such as dracaena, Moraceae suchas ficus, Araceae such as sweetheart plant, and others.

Most preference is given to Arabidopsis thaliana, Nicotiana tabacum,Tagetes erecta, Calendula officinalis and Brassica napus and to allgenera and species which are used as food- or feedstuffs, such as thecereal species described, or which are suitable for preparing oils, suchas oilseeds (e.g. oilseed rape), nut species, soya, sunflower, pumpkinand peanut.

Plant organisms for the purposes of this invention are furthermore otherorganisms capable of photosynthetic activity, such as, for example,algae or cyanobacteria, and also mosses. Preferred algae are green algaesuch as, for example, algae of the genus Haematococcus, Phaedactylumtricornatum, Volvox or Dunaliella.

The preparation of a transformed organism or of a transformed cellrequires introducing the appropriate DNA into the appropriate host cell.A multiplicity of methods is available for this process which isreferred to as transformation (see also Keown et al. 1990 Methods inEnzymology 185:527-537). Thus, by way of example, the DNA may beintroduced directly by microinjection or by bombardment with DNA-coatedmicroparticles. The cell may also be permeabilized chemically, forexample using polyethylene glycol, so that the DNA can enter the cellvia diffusion. The DNA may also be formed via protoplast fusion withother DNA-comprising units such as minicells, cells, lysosomes orliposomes. Another suitable method for introducing DNA iselectroporation in which the cells are reversibly permeabilized by anelectric impulse.

In the case of plants, the methods described for transforming andregenerating plants from plant tissues or plant cells are utilized fortransient or stable transformation. Suitable methods are especiallyprotoplast transformation by polyethylene glycol-induced DNA uptake, thebiolistic method using the gene gun, the “particle bombardment” method,electroporation, the incubation of dry embryos in DNA-comprisingsolution and microinjection.

Apart from these “direct” transformation techniques, a transformationmay also be carried out by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes. These strains contain a plasmid(Ti or R1 plasmid) which is transferred to the plant after Agrobacteriainfection. A part of this plasmid, denoted T-DNA (transferred DNA) isintegrated into the genome of the plant cell.

The Agrobacterium-mediated transformation is best suited todicotyledonous diploid plant cells, whereas the direct transformationtechniques are suitable for any cell type.

An expression cassette of the invention may be introduced advantageouslyinto cells, preferably into plant cells, by using vectors.

In an advantageous embodiment, the expression cassette is introduced bymeans of plasmid vectors. Preference is given to those vectors whichenable a stable integration of the expression cassette into the hostgenome.

In the case of injection or electroporation of DNA into plant cells, noparticular demands on the plasmid used are made. It is possible to usesimple plasmids such as those of the pUC series. If complete plants areto be regenerated from the transformed cells, it is necessary for anadditional selectable marker gene to be present on the plasmid.

Transformation techniques have been described for variousmonocotyledonous and dicotyledonous plant organisms. Furthermore,various possible plasmid vectors which normally contain an origin ofreplication for propagation in E. coli and a marker gene for selectionof transformed bacteria are available for introducing foreign genes intoplants. Examples are pBR322, pUC series, M13 mp series, pACYC184 etc.

The expression cassette may be introduced into the vector via a suitablerestriction cleavage site. The resultant plasmid is first introducedinto E. coli. Correctly transformed E. coli cells are selected,cultivated and the recombinant plasmid is obtained using methodsfamiliar to the skilled worker. Restriction analysis and sequencing maybe used in order to check the cloning step.

Transformed cells, i.e. those which contain the introduced DNAintegrated into the DNA of the host cell may be selected fromuntransformed cells, if a selectable marker is part of the introducedDNA. A marker may be, by way of example, any gene which is capable ofimparting a resistance to antibiotics or herbicides. Transformed cellswhich express such a marker gene are capable of surviving in thepresence of concentrations of an appropriate antibiotic or herbicide,which kill an untransformed wild type. Examples are the bar gene whichimparts resistance to the herbicide phosphinothricin (Rathore K S etal., Plant Mol. Biol. 1993 March; 21(5):871-884), the nptII gene whichimparts resistance to kanamycin, the hpt gene which imparts resistanceto hygromycin and the EPSP gene which imparts resistance to theherbicide glyphosate.

Depending on the method of DNA introduction, further genes may berequired on the vector plasmid. If agrobacteria are used, the expressioncassette is to be integrated into specific plasmids, either into anintermediate vector (shuttle vector) or a binary vector. If, forexample, a Ti or Ri plasmid is to be used for transformation, at leastthe right border, in most cases, however, the right and the left border,of the Ti or Ri plasmid T-DNA is connected as flanking region with theexpression cassette to be introduced. Preference is given to usingbinary vectors. Binary vectors can replicate both in E. coli and inAgrobacterium. They normally contain a selection marker gene and alinker or polylinker flanked by the right and left T-DNA bordersequences. They may be transformed directly into Agrobacterium (Holsterset al., Mol. Gen. Genet. 163 (1978), 181-187). The selection marker genepermits selection of transformed Agrobacteria; an example is the nptIIgene which imparts a resistance to kanamycin. The Agrobacterium which inthis case acts as the host organism should already contain a plasmidwith the vir region. This region is required for the transfer of T-DNAinto the plant cell. An Agrobacterium transformed in this way may beused for transformation of plant cells.

The use of T-DNA for transformation of plant cells has been intenselystudied and described (EP 120516; Hoekema, In: The Binary Plant VectorSystem, Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; Fraleyet al., Crit. Rev. Plant. Sci., 4:1-46 and An et al., EMBO J. 4 (1985),277-287). Various binary vectors are known and partly commerciallyavailable, such as, for example, pBIN19 (Clontech Laboratories, Inc.U.S.A.).

The DNA is transferred into the plant cell by coculturing plant explantswith Agrobacterium tumefaciens or Agrobacterium rhizogenes. Startingfrom infected plant material (e.g. leaf, root or stem parts, but alsoprotoplasts or plant cell suspensions), it is possible to regeneratewhole plants by using a suitable medium which may contain, for example,antibiotics or biocides for selection of transformed cells. The plantsobtained may then be screened for the presence of the introduced DNA, inthis case the expression cassette of the invention. As soon as the DNAhas integrated into the host genome, the corresponding genotype isnormally stable and the corresponding insertion is also found again insubsequent generations. Normally, the integrated expression cassettecontains a selection marker which imparts to the transformed plant aresistance to a biocide (for example a herbicide), a metabolisminhibitor such as 2-DOG or an antibiotic such as kanamycin, G 418,bleomycin, hygromycin or phosphinothricin etc. The selection markerallows the selection of transformed cells from untransformed cells(McCormick et al., (1986) Plant Cell Reports 5: 81-84). The plantsobtained may be cultivated and crossed in the common manner. Two or moregenerations should be cultured in order to ensure that the genomicintegration is stable and heritable.

The abovementioned methods are described, for example, in B. Jenes etal., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by S. D. Kung and R. Wu, AcademicPress (1993), pp. 128-143 and in Potrykus, (1991) Annu Rev. PlantPhysiol. Plant Molec. Biol. 42: 205-225). The construct to be expressedis preferably cloned into a vector which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., (1984)Nucl. Acids Res. 12: 871 f.).

As soon as a transformed plant cell has been prepared, it is possible toobtain a complete plant by using methods known to the skilled worker. Tothis end, callus cultures are used as starting point, by way of example.From these still undifferentiated cell masses, it is possible to induceformation of shoot and root in the known manner. The shoots obtained canbe planted out and cultivated.

The efficacy of expression of the nucleic acids to be expressedtransgenically can be determined, for example, in vitro by shootmeristem propagation using one of the above-described selection methods.

The invention further relates to cells, cell cultures, parts, such as,for example, roots, leaves, etc. in the case of transgenic plantorganisms, and transgenic propagation material such as seeds or fruitsderived from the above-described transgenic organisms.

Genetically modified plants of the invention, which can be consumed byhumans and animals, may also be used, for example directly or afterpreparation known per se, as foodstuffs or feedstuffs.

The invention further relates to the use of the above-describedtransgenic organisms of the invention and of the cells, cell cultures,parts, such as, for example, roots, leaves, etc., in the case oftransgenic plant organisms, and transgenic propagation material such asseeds or fruits for the production of food- or feedstuffs,pharmaceuticals or fine chemicals.

Preference is further given to a method for the recombinant productionof pharmaceuticals or fine chemicals in host organisms, in which a hostorganism is transformed with one of the above-described expressioncassettes or vectors and said expression cassette contains one or morestructural genes which code for the fine chemical of interest orcatalyze the biosynthesis of the fine chemical of interest, and thetransformed host organism is cultivated and the fine chemical ofinterest is isolated from the cultivation medium. This method is broadlyapplicable for fine chemicals such as enzymes, vitamins, amino acids,sugars, fatty acids, natural and synthetic flavorings, aromatizingsubstances and colorants. Particular preference is given to theproduction of tocopherols and tocotrienols and also carotenoids.Cultivation of the transformed host organisms and isolation from saidhost organisms or from the cultivation medium are carried out by meansof the methods known to the skilled worker. The production ofpharmaceuticals such as, for example, antibodies or vaccines isdescribed in Hood E E, Jilka J M (1999). Curr Opin Biotechnol.10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol.236:275-92.

Sequences:

SEQ ID NO: 1 Promoter and 5′-untranslated region of theArabidopsis thaliana pFD promoter. SEQ ID NO: 2Promoter and 5′-untranslated region of theArabidopsis thaliana FNR promoter. SEQ ID NO: 3Promoter and 5′-untranslated region of theArabidopsis thaliana TPT promoter (2038 bp). SEQ ID NO: 4Promoter and 5′-untranslated region of thetruncated Arabidopsis thaliana pFDs promoter. SEQ ID NO: 5Nucleic acids coding for a phosphinothricin acetyltransferase.SEQ ID NO: 6 Amino acid sequence coding for a phosphinothricinacetyltransferase. SEQ ID NO: 7Nucleic acid coding for an acetolactate synthase. SEQ ID NO: 8Amino acid sequence coding for an acetolacate synthase.SEQ ID NO: 9-oligonucleotide primer pWL355′-GTC GAC GAA TTC GAG AGA CAG AGA GAC GG-3′SEQ ID NO: 10-oligonucleotide primer pWL365′-GTC GAC GGT ACC GAT TCA AGC TTC ACT GC-3′SEQ ID NO: 11-oligonucleotide primer pFD15′-GAG AAT TCG ATT CAA GCT TCA CTG C-3′SEQ ID NO: 12-oligonucleotide primer pFD25′-CCA TGG GAG AGA CAG AGA GAC G-3′SEQ ID NO: 13-oligonucleotide primer pFD35′-acggatccgagagacagagagacggagacaaaa-3′SEQ ID NO: 14-oligonucleotide primer pFD55′-gcggatccaacactcttaacaccaaatcaaca-3′SEQ ID NO: 15-oligonucleotide primer L-FNR ara5′-GTCGACGGATCCGGTTGATCAGAAGAAGAAGAAGAAG ATGAACT-3′SEQ ID NO: 16-oligonucleotide primer R-FNR ara5′-GTCGACTCTAGATTCATTATTTCGATTTTGATTTCGT GACC-3′SEQ ID NO: 17-oligonucleotide primer L-TPT ara5′-AGTCGACGGATCCATAACCAAAAGAACTCTGATCATGT ACGTACCCATT-3′SEQ ID NO: 18-oligonucleotide primer R-TPT ara5′-AGACGTCGACTCTAGATGAAATCGAAATTCAGAGT TTTGA TAGTGAGAGC-3′SEQ ID NO: 19-oligonucleotide primer ubi55′-CCAAACCATGGTAAGTTTGTCTAAAGCTTA-3′SEQ ID NO: 20-oligonucleotide primer ubi35′-CGGATCCTTTTGTGTTTCGTCTTCTCTCA-CG-3′SEQ ID NO: 21-oligonucleotide primer sqs55′-GTCTAGAGGCAAACCACCGAGTGTT-3′SEQ ID NO: 22. oligonucleotide primer sqs35′-CGGTACCTGTTTCCAGAAAATTTTGATTCAG-3′ SEQ ID NO: 23binary plasmid pSUN3 (Sungene GmbH & Co KGaA) SEQ ID NO: 24binary plasmid pSUN5NPTIICat (Sungene GmbH & Co KGaA) SEQ ID NO: 25binary plasmid pSUN3PatNos (Sungene GmbH & Co KGaA)SEQ ID NO: 26-oligonucleotide primer 5-TPTara5′-AAG TCG ACG GAT CCT GAT AGC TTA TAC TCA AAT TCA ACA AGT TAT-3′SEQ ID NO: 27 truncated promoter and 5′-untranslated region ofthe Arabidopsis thaliana and TPT-Promoters (1318 bp) SEQ ID NO: 28nucleic acid sequence of the terminator of thepotato cathepsin D inhibitor gene (GenBank Acc. No. X74985)SEQ ID NO: 29 nucleic acid sequence of the terminator of thefield bean storage protein gene VfLE1B3 (GenBank Acc. No. Z26489).

DESCRIPTION OF THE FIGURES

FIG. 1 a-c: The TPT and the FNR promoters show a comparable expressionpattern in green tissue and in flowers of tobacco and potato.GUS-histochemical stains are formed. The intensity of the GUS blue staincorresponds to the shades of gray displayed. The figures show:

In FIG. 1 a:

A: Potato leaves with a homogeneous intensity stain over the entire leafregion.

B: Tobacco petioles, intensive blue stain, especially on the edges andin the vascular regions (see arrow).

In FIG. 1 b:

C: Tobacco stems, intensive blue stains, especially on the edges (seearrow). D: Tobacco internodia.

In FIG. 1 c:

E: Tobacco flower; blue stain, especially in sepals and petals.

FIG. 2 a-b: The TPT promoter and the FNR promoter show a differentexpression pattern in vegetative and germinative storage tissue oftobacco and potato. While the TPT promoter is active here, the FNRpromoter shows no expression. GUS histochemical stains of tobacco seedsand tobacco seedlings and also of potato tubers are shown. However, bothpromoters exhibit again a comparable activity in seedlings. Theintensity of the GUS blue stain corresponds to the shades of graydisplayed. The figures show:

In FIG. 2 a:

A: Tobacco seeds. In the case of the TPT promoter, individual bluestained seeds are visible (see arrow). In the case of the FNR promoter,no stains are detectable.

B: Potato tubers. In the case of the TPT promoter, a homogenous strongblue stain of the potato tuber is visible. In the case of the FNRpromoter, only a very weak stain is detectable, if at all.

In FIG. 2 b:

C: Tobacco seedlings (10 days old). Both promoters show a comparableblue stain (see arrow).

Expression cassettes for the expression of kanamycin-resistance (nptII)and phosphinothricin-resistance (pat) markers. Cassette A permitsexpression of kanamycin resistance under the TPT or FNR promoter, inaddition to a phosphinothricin resistance under the NOS promoter.Cassette B permits expression of phosphinothricin resistance under theTPT or FNR promoter, in addition to kanamycin resistance under the NOSpromoter.

LB, RB: left and right border, respectively, of Agrobacterium T-DNA

nosP: NOS promoter

pat: nucleic acid sequence coding for phosphinothricin acetyltransferase(pat)

nptII: kanamycin resistance gene (Neomycin phosphotransferase)

nosT: NOS terminator

FNR-P: FNR promoter

TPT-P: TPT promoter

Regeneration of transformed tobacco plumulae under kanamycin selectionpressure (100 mg/l kanamycin). A: transformation with an FNRpromoter-nptII construct. B: transformation with a TPT promoter-nptIIconstruct. A comparable efficient regeneration of transformed tobaccoplants was observed.

Germination of transformed tobacco plants from transgenic tobacco seedsunder phosphinothricin selection pressure (10 mg/l phosphinotricin).

A: transformed with an FNR promoter-pat construct.

B: transformed with a TPT promoter-pat construct.

C: control with untransformed tobacco seeds.

A comparably efficient germination of tobacco plants transformed withthe FNR promoter-pat construct and the TPT promoter-pat construct wasobserved, while untransformed tobacco plants treated in a correspondingmanner had no resistance.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention.

EXAMPLES General Methods

The chemical synthesis of oligonucleotides may be carried out in amanner known per se, for example according to the phosphoramidite method(Voet, 2^(nd) edition, Wiley Press New York, pages 896-897). The cloningsteps carried out within the framework of the present invention, suchas, for example, restriction cleavages, agarose gel electrophoreses,purification of DNA fragments, transfer of nucleic acids tonitrocellulose and nylon membranes, ligation of DNA fragments,transformation of E. coli cells, cultivation of bacteria, propagation ofphages and sequence analysis of recombinant DNA, are carried out asdescribed in Sambrook et al. (1989) Cold Spring Harbor Laboratory Press;ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced according tothe method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA74:5463-5467), using a laser fluorescence DNA sequencer from ABI.

Example 1 Isolation of Genomic DNA from Arabidopsis thaliana (CTABMethod)

Genomic DNA is isolated from Arabidopsis thaliana by grinding approx.0.25 g of leaf material of young plants in the vegetative state inliquid nitrogen to give a fine powder. The pulverulent plant material isintroduced together with 1 ml of 65° C. CTAB I buffer (CTAB:hexadecyltrimethylammonium bromide, also called cetyltrimethylammoniumbromide; Sigma Cat.-No.: H6269) and 20 μl of β-mercaptoethanol into aprewarmed second mortar and, after complete homogenization, the extractis transferred to a 2 ml Eppendorf vessel and incubated with carefulregular mixing at 65° C. for 1 h. After cooling to room temperature, themixture is extracted with 1 ml of chloroform/octanol (24:1, equilibratedby shaking with 1 M Tris/HCl, pH8.0) by slowly inverting the vessel andthe phases are separated by centrifugation at 8,500 rpm (7,500×g) androom temperature for 5 min. Subsequently, the aqueous phase is extractedagain with 1 ml of chloroform/octanol, centrifuged and carefully mixedwith 1/10 volume of CTAB II buffer prewarmed to 65° C. by inverting thevessel. One ml of chloroform/octanol mixture (see above) is then addedwith careful agitation to the reaction mixture and the phases are againseparated by centrifugation at 8,500 rpm (7,500×g) and room temperaturefor 5 min. The aqueous lower phase is transferred to a fresh Eppendorfvessel and the upper organic phase is again centrifuged in a freshEppendorf vessel at 8,500 rpm (7,500×g) and room temperature for 15 min.The aqueous phase resulting herefrom is combined with the aqueous phaseof the previous centrifugation step and the entire reaction mixture isthen mixed with exactly the same volume of prewarmed CTAB III buffer.This is followed by an incubation at 65° C. until the DNA precipitatesin flakes. This may continue for up to 1 h or be affected by incubationat 37° C. overnight. The sediment resulting from the subsequentcentrifugation step (5 min, 2000 rpm (500×g), 4.degree° is admixed with250 μl of CTAB IV buffer prewarmed to 65.degree. C., and the mixture isincubated at 65° C. for at least 30 min or until the sediment hascompletely dissolved. The DNA is then precipitated by mixing thesolution with 2.5 volumes of ice-cold ethanol and incubating at −20° C.for 1 h. As an alternative, the reaction mixture is mixed with 0.6volumes of isopropanol and, without further incubation, immediatelycentrifuged at 8,500 rpm (7,500×g) and 4° C. for 15 min. The sedimentedDNA is washed twice with in each case 1 ml of 80% strength ice-coldethanol by inverting the Eppendorf vessel, each washing step beingfollowed by another centrifugation (5 min, 8,500 rpm (7,500×g), 4° C.)and the DNA pellet is then dried in air for approx. 15 min Finally, theDNA is resuspended in 100 μl of TE comprising 100 μg/ml RNase and themixture is incubated at room temperature for 30 min. After anotherincubation phase at 4° C. overnight, the DNA solution is homogeneous andcan be used for subsequent experiments.

Solution for CTAB:

Solution I (for 200 ml):

100 mM Tris/HCl pH 8.0 (2.42 g)

1.4 M NaCl (16.36 g)

20 mM EDTA (8.0 ml of 0.5 M stock solution)

2% (w/v) CTAB (4.0 g)

The following is added in each case prior to use: 2% β-mercaptoethanol(20 μl for 1 ml of solution I).

Solution II (for 200 ml):

0.7 MNaCl (8.18 g)

10% (w/v) CTAB (20 g)

Solution III (for 200 ml):

50 mM Tris/HCl pH 8.0 (1.21 g)

10 mM EDTA (4 ml 0.5 M of 0.5 M stock solution)

1% (w/v) CTAB (2.0 g)

Solution IV (High-Salt TE) (for 200 ml):

10 mM Tris/HCl pH 8.0 (0.242 g)

0.1 mM EDTA (40 μl of 0.5 M stock solution)

1 M NaCl (11.69 g)

Chloroform/Octanol (24:1) (for 200 ml):

192 ml of chloroform

8 ml of octanol

The mixture is equilibrated by shaking 2× with 1 M Tris/HCl pH 8.0 andstored protected from light.

Example 2 Transformation of Tobacco, Oilseed Rape and Potato

Tobacco was transformed via infection with Agrobacterium tumefaciens.According to the method developed by Horsch (Horsch et al. (1985)Science 227: 1229-1231). All constructs used for transformation weretransformed into Agrobacterium tumefaciens by using the freeze/thawmethod (repeated thawing and freezing). The Agrobacterium coloniescomprising the desired construct were selected on mannitol/glutamatemedium comprising 50 μg/ml kanamycin, 50 μg/ml ampicillin and 25 μg/mlrifampicin.

Tobacco plants (Nicotiana tabacum L. cv. Samsun N N) were transformed bycentrifuging 10 ml of an Agrobacterium tumefaciens overnight culturegrown under selection, discarding the supernatant and resuspending thebacteria in the same volume of antibiotics-free medium. Leaf disks ofsterile plants (approx. 1 cm in diameter) were bathed in this bacteriasolution in a sterile Petri dish. The leaf disks were then laid out inPetri dishes on MS medium (Murashige and Skoog (1962) Physiol Plant15:473ff.) comprising 2% sucrose and 0.8% Bacto agar. After incubationin the dark at 25° C. for 2 days, they were transferred to MS mediumcomprising 100 mg/l kanamycin, 500 mg/i Claforan, 1 mg/lbenzylaminopurine (BAP), 0.2 mg/l naphthylacetic acid (NAA), 1.6%glucose and 0.8% Bacto agar and cultivation was continued (16 hourslight/8 hours dark). Growing shoots were transferred to hormone-free MSmedium comprising 2% sucrose, 250 mg/l Claforan and 0.8% Bacto agar.

Oilseed rape was transformed by means of petiole transformationaccording to Moloney et al. (Moloney M M, Walker J M & Sharma K K (1989)Plant Cell Reports 8:238-242).

Potatoes (Solanum tuberosum) were transformed by infecting leaf disksand internodia of in vitro plants with Agrobacterium tumefaciens inliquid Murashige Skoog Medium for 20 minutes and then coculturing themin the dark for 2 d. After coculturing, the explants were cultured onsolid MS medium which contains instead of sucrose 1.6% glucose (MG) andwhich has been supplemented with 5 mg/l NAA, 0.1 mg/l BAP, 250 mg/lTimentin and 30 to 40 mg/l kanamycin (KIM), at 21° C. in a 16 h light/8h dark rhythm. After this callus phase, the explants were placed onshoot induction medium (SIM). SIM was composed as follows: MG+2 mg/lZeatinriboside, 0.02 mg/l NAA, 0.02 mg/l GA3, 250 mg/l Timentin, 30 to40 mg/l kanamycin. Every two weeks, the explants were transferred tofresh SIM. The developing shoots were rooted on MS medium comprising 2%sucrose and 250 mg/l Timentin and 30 to 40 mg/l kanamycin.

Example 3 Studies on the Suitability of the Putative Ferredoxin (pFD)Promoter

Cloning of the pFD Promoters from Arabidopsis thaliana

The putative ferredoxin promoter was amplified from genomic Arabidopsisthaliana DNA by means of PCR using the primers pWL35 and pWL36. Theprimer pWL35 starts with the SalI and EcoRI restriction cleavage siteswhich are located immediately upstream of the coding region of the pFDgene and are highlighted in bold type. The primer pWL36 starts with theSalI and Asp718 restriction cleavage sites highlighted in bold type.

Primer pWL35 (SEQ ID NO: 9) 5′ GTC GAC GAA TTC GAG AGA CAG AGA GAC GG 3′Primer pWL36 (SEQ ID NO: 10) 5′GTC GAC GGT ACC GAT TCA AGC TTC ACT GC 3′

Reaction Mixture:

-   -   1 μl Genomic Arabidopsis DNA (approx. 250 ng)    -   0.5 μl Tth polymerase (2 U/.mu.l)    -   3 μl Mg(OAc)₂ (25 mM, final conc. 1.5 mM Mg²⁺)    -   15.2 μl 3.3× buffer    -   4 μl dNTPs (2.5 mM each, Takara, final concentration: 200 μM        each)    -   24.3 μl H₂O

PCR Conditions:

-   -   1 cycle at 95° C. for 3 min    -   10 cycles at 94° C. for 10 s, 50° C. for 20 s and 72° C. for 1        min    -   20 cycles at 94° C. for 10 s, 65° C. for 20 s and 72° C. for 1        min    -   1 cycle at 72° C. for 5 min.—followed by cooling to 4° C. until        further use.

b) Construction of the pFD Promoter-GUS Expression Cassette.

The PCR product of the pFD promoter was cloned into the pCRII vector(Invitrogen) and subsequently isolated by means of the SalI restrictioncleavage sites introduced by the pair of primers and purified by gelelectrophoresis. For fusion with the GUS gene, the approx. 850 bp pFDpromoter fragment was cloned into the Sail-cut binary vector pBI101.2(Clontech Inc.) and the orientation of the fragment was subsequentlyverified on the basis of restriction analyses using the endonucleasesBglII and BamHI. The resulting plasmid pFD::GUS was transformed intotobacco. The tobacco plants generated were denoted pFD:GUS.

c) Construction of the pFD Promoter-NptII Expression Cassette.

The putative ferredoxin promoter was amplified from genomic Arabidopsisthaliana DNA by means of PCR. The primers were used to add therestriction sites EcoRI and NcoI.

Primer pFD1 (SEQ ID NO: 11) 5′ GAG AAT TCG ATT CAA GCT TCA CTG CPrimer pFD2 (SEQ ID NO: 12) 5′ CCA TGG GAG AGA CAG AGA GAC G

Reaction Mixture:

-   -   37.5 μl H₂O    -   5.0 μl 10× reaction buffer (final concentration Mg²⁺ 1.5 mM)    -   4.0 μl dNTP mix (2.5 mM each)    -   1.0 μl Primer pFD1 (10 μM)    -   1.0 μl Primer pFD2 (10 μM)    -   0.5 μl Taq polymerase (Takara, 2 U/μl)    -   1.0 μl genomic Arabidopsis DNA (approx. 250 ng)

PCR Conditions:

-   -   cycle at 94° C. for 3 min    -   cycles at 94° C. for 10 s, 48° C. for 20 s and 72° C. for 1 min.    -   cycles at 94° C. for 10 s, 65° C. for 20 s and 72° C. for 1 min.    -   cycle at 72° C. for 5 min.

The PCR product was subcloned into the pCRII plasmid (Invitrogen). Theplasmid pCAMBIA 2300 (CAMBIA, GPO Box 3200, Canberra ACT 2601,Australia; GenBank Acc. No: AF234315; Binary vector pCAMBIA-2300,complete sequence; Hajdukiewicz P et al. (1994) Plant Mol Biol25(6):989-994) was cut with EcoRI/NcoI and the pFD promoter fragment wascloned as EcoRI/NcoI fragment from the pCRII plasmid into this vector.In the process, the 35S promoter was removed from the Cambia vector. Theresulting plasmid was referred to as pFD promoter:NPTII and transformedinto tobacco.

Results of GUS Analysis of the Transgenic Tobacco Plants.

In the context of histochemical investigations, transgenic pFD::GUStobacco plants showed strong GUS staining in source leaves and weak GUSstaining in the tissues of all flower organs. Strong staining in roottissue was only observed in in vitro plants whose roots had been exposedto the illumination. Callus growth was induced on the basis of leafdisks which had been punched out of plants identified aspFD::GUS-positive. The callus tissue and also the plant shootsdeveloping therefrom showed GUS staining whose intensity was comparableto that of the GUS staining of CaMV35S::GUS (in pCambia 1304; CAMBIA,GPO Box 3200, Canberra ACT 2601, Australia; GenBank Acc. No.: AF234300,Binary vector pCAMBIA-1304, complete sequence, Hajdukiewicz P et al.(1994) Plant Mol. Biol. 25(6):989-994) (1994)) transgenic plants. Thetable listed below (Table 3) summarizes the data of quantifying the GUSactivity in the anthers and source leaves of selected transgenicpFD::GUS tobacco plants.

TABLE 3 Quantification of GUS activity in anthers and source leaves ofselected transgenic pFD::GUS tobacco plants. GUS-Activity (pmol[4MU]/mg[protein]/min) pFD::GUS Plant no. Anthers ‘Source’ leaves pFD5275 3785 pFD11 174 6202 pFD14 362 2898 pFD15 57 2678

The anthers of the mature flowers display no promoter activity. Saidactivity is weak in closed flowers.

e) Results of the Analysis of Kanamycin Resistance of the TransgenicTobacco Plants.

In order to study the pFD promoter-assisted imparting of resistance tokanamycin, the pFD promoter:NPTII plasmid was transformed into tobacco.The tobacco plants were selectively regenerated on kanamycin (100 mg/l).The plants regenerated from the developing plumulae comprised kanamycin,demonstrating that the pFD promoter had expressed the NPTII gene andthus made selection possible. The results demonstrate that the isolatednucleic acid sequence has the desired advantageous promoter properties,i.e. it exhibits a promoter activity which is suitable for expressingselection markers effectively and its activity in the pollen is low. Theactivity in the anthers is normally less than 10% of the activity in thesource leaves.

f) Results of GUS Analysis of the Transgenic Potato Plants.

The pFD:GUS plasmid (cf. Example 3b) is transformed into potatoesaccording to the method described in Example 2.

Result of functional studies: The pFD promoter is strongly expressed inthe leaves of the transgenic potato plants analyzed. GUS staining wasfound to be stronger in the leaves of the potato plants than in theleaves of the tobacco plants described. Weak staining of the flowers andno staining of the tubers indicated low expression in the flowers and noexpression in the tubers, respectively.

The data demonstrate that this promoter has no activity in the tubers ofpotato plants and is suitable for the expression of genes, for exampleof insecticides, in the leaves and other organs above the ground ofplants, whose gene products are unwanted in the storage organs.

Preparation of Deletion Variants of the pFD Promoter.

A further pFD promoter variant is the deletion pFD-short (pFds). Forthis purpose, the pFD promoter section from base pairs 137 to 837 wasamplified using the following primers:

pFD3 (SEQ ID NO: 13): 5′-acggatccgagagacagagagacggagacaa-aa-3′:pFD5 (SEQ ID NO: 14): 5′gcggatccaacactataacaccaaatcaaca-3′:

Reaction Mixture:

-   -   37.5 μl H₂O    -   5.0 μl 10× reaction buffer (“genomic PCR”)    -   4.0 μl dNTP mix (2.5 mM each)    -   2.2 μl 25 mM Mg(OAc).sub.2 (final concentration 1.1 mM)    -   1.0 μl Primer pFD3 (10 μM)    -   1.0 μl Primer pFD5 (10 μM)    -   0.5 μl Pfu-turbo polymerase mix    -   1.0 μl Genomic Arabidopsis DNA (approx. 250 ng)

PCR Conditions:

-   -   cycle at 95° C. for 5 min    -   25 cycles at 94° C. for 30 s, 50° C. for 60 s and 72° C. for 1        min.    -   cycle at 50° C. for 60 s, 72° C. for 10 min, followed by cooling        to 4° C. until further use.

The primers comprised recognition sequences for the restriction enzymeBamHI. After BamHI cleavage, the PCR product was ligated into theplasmid pGUSINT37 (see above) which had likewise been cut with BamHI andhad been dephosphorylated. Tobacco leaves were bombarded with theresulting construct pFDsGUSINT by means of Biolistics (BioRad). In thisconnection, microcarriers (25 μg of Gold, Heraeus 0.3 to 3 μm) weretreated with 10 μg of plasmid DNA, 2.5 M CaCl₂, and 0.1 M spermidine,washed with alcohol and fired at the leaves which were lying on MSmedium under a vacuum of 26 inches and a pressure of 1100 psi. Theexplants were then incubated in MS medium comprising 2% sucrose for 24 hand then histochemically stained with X-gluc. Blue spots indicated theactivity of the promoter.

Fusing the pFDs Promoter to the NPTII Gene.

The pFDs promoter is excised as BamHI fragment from pFDsGUSINT and itsends are rendered blunt by means of Klenow-“Fill-In.” The fragmentobtained is cloned upstream of the NPTII gene of the EcoRV-cut anddephosphorylated plasmid pSUN5NPTIICat (SEQ ID NO: 24). The plasmidpSUN5NPTII is a derivative of plasmid pSUN3 (SEQ ID NO: 23), whichcontains, apart from nosP/Pat cassette, also a promoterless NPTII gene.This construct makes it possible to assay promoters on their ability toexpress NPTII. Selection on phosphinothricin-comprising medium may becarried out in parallel.

The resulting plasmid pSun5FdsNPTII is transformed into tobacco.Regenerated and selected shoots showed that the pFDs promoter allowsselection for NPTII.

Example 4 Studies on the Suitability of the Ferredoxin NADPHOxidoreductase (FNR) Promoter

Cloning of the FNR Promoter from Arabidopsis thaliana.

The putative promoter region of the FNR gene was amplified from genomicDNA by using the oligonucleotide primers L-FNRara and R-FNRara,bypassing the ATG start codon of the FNR gene and retaining fourputative stop codons of the open reading frame located upstream. Usingthe primers L-FNRara and R-FNRara, the FNR promoter was amplified as a635 bp fragment corresponding to the section of the clone K2A18.15 fromposition 69493 to position 70127 (including these two nucleotides) fromgenomic Arabidopsis thaliana DNA by means of PCR. The primer L-FNRarastarts with the restriction cleavage sites SalI and BamHI highlighted inbold type and is located upstream of the four stop codons of the genelocated upstream of the FNR promoter. The primer R-FNRara starts withthe SalI and XbaI restriction cleavage sites which are locatedimmediately upstream of the ATG start codon of the FNR gene and arehighlighted in bold type.

Primer L-FNR ara (44 mer) (SEQ ID NO: 15): 5′GTC GAC GGA TCC GGT TGA TCA GAA GAA GAA GAA GAA GAT GAA CT 3′Primer R-FNR ara (41 mer) (SEQ ID NO: 16): 5′GTC GAC TCT AGA TTC ATT ATT TCG ATT TTG ATT TCG TGA CC 3′

The FNR promoter was amplified using a “touchdown” PCR protocol with theuse of the ‘Advantage Genomic Polymerase Mix’ (Clontech Laboratories,Inc; Catalogue No. #8418-1). The above-mentioned polymerase mix containsa thermostable DNA polymerase from Thermus thermophilus (Tth DNApolymerase), mixed with a smaller proportion of Vent proofreading 3′-5′polymerase, and the Tth start antibody which makes hot-start PCRpossible.

Reaction Mixture:

-   -   36.8 μl H₂O    -   5 μl 10× reaction buffer (“genomic PCR”)    -   1 μl dNTP mix (10 mM each)    -   2.2 μl 25 mM Mg(OAc)² (final concentration 1.1 mM)    -   1 μl Primer L-FNR ara (10 μM)    -   1 μl Primer R-FNR ara (10 μM)    -   1 μl 50× polymerase mix    -   2 μl Genomic Arabidopsis DNA (approx. 500 ng)

PCR Conditions:

-   -   1 cycle at 94° C. for 1 min    -   10 cycles at 94° C. for 30 s and 70° C. for 3 min.    -   32 cycles at 94° C. for 30 s and 65° C. for 3 min.    -   1 cycle at 65° C. for 4 min.—followed by cooling to 4° C. until        further use.

Construction of the FNR Promoter-GUS Expression Cassette.

After gel-electrophoretic fractionation and purification from the gelusing the Quiagen PCR purification kit, the PCR product of the FNRpromoter was cloned into the pCRII vector (Invitrogen) via TA cloningThe promoter fragment was then isolated from the resulting plasmidpATFNR1 bp digestion with XbaI/BamHI by means of the XbaI and BamHIrestriction cleavage sites introduced by the pair of primers andpurified by gel electrophoresis. For fusion with the GUS gene, theapprox. 600 bp FNR promoter fragment was cloned into theXbaI/BamHI-digested binary vector pBI101. The correct insertion of thecorrect fragment in the resulting plasmid pATFNR-Bi was then verified onthe basis of a restriction analysis using the endonuclease EcoRV. Theplasmid pATFNR-Bi was used for transformation of tobacco.

For transformation in oilseed rape, the FNR promoter was cloned as SalIfragment of plasmid pCR_ATFNR into the vector pS3NitGUS cut with SalIand XhoI, thereby replacing the nitrilase promoter.

Construction of the FNR Promoter-PAT Expression Cassette.

In order to study the FNR promoter-assisted imparting of resistance tophosphinothricin, the FNR promoter was cloned as SalI fragment fromplasmid pATFNR1 into the SalI-cut plasmid pSUN3 PatNos (SEQ ID NO: 25)upstream of the phosphinothricin resistance gene.

Construction of the FNR Promoter-NptII Expression Cassette.

In order to impart resistance to kanamycin, the FNR promoter was clonedas SalI fragment into the Xha-cut dephosphorylated plasmid pSUN5NptIICat(Sungene GmbH & Co KGaA, SEQ ID NO: 24) upstream of the NPTII resistancegene. The resulting plasmid is referred to as pS5FNRNptII and wastransformed into tobacco and oilseed rape.

Results of GUS Analysis of the Transgenic Tobacco Plants.

Qualitative data: Transgenic FNR::GUS-Tobacco plants displayed strongGUS expression in all green tissues, especially in source leaves, leafstalks and internodia, and also in all flower organs of fully developedflowers, (ovary, stigma, sepals and petals) with the exception of pollenwhich showed no GUS activity; a low staining intensity was detected inanthers. In the first analysis of leaf disks of 80 in vitro plants, 70plants displayed strong GUS staining with low variation in stainingintensity between the individual plants. This was regarded as anindication that the FNR promoter contains an element which provideslimited positional effects. In the tissue culture plants, the GUSactivity of the FNR::GUS plants was markedly lower than the activity ofTPT::GUS plants. Transgenic oilseed rape plants displayed the samestaining pattern.

Seed material (F1) of the lines FNR 13, FNR 45 and FNR 28 was analyzedwith respect to its GUS activity. It turned out that GUS activity wasdetected neither in resting seeds nor in growing seedlings (3.5 daysafter sowing).

In later seedlings stages (6 and 10 days after sowing), strong GUSactivity was detected in the cotyledons and in the upper region of theseedling axis, whereas no GUS staining was detected in the roots. Inseedlings which had been cultivated in the dark, GUS activity waslimited to the cotyledons and was overall lower than in thelight-germinated seedlings.

Quantitative analysis of the GUS activity in FNR::GUS transgenic tobaccoplants (transformed with plasmid pATFNR-Bi) was analyzed on the firstfully developed leaves of tobacco plants 21 days after transfer from thetissue culture to the greenhouse. The data corresponds to the average offour independent measurements.

TABLE 4 Quantification of GUS activity in leaf material of selectedtransgenic FNR::GUS tobacco plants (transformed with plasmid pATFNR-Bi).Rank (x-strongest GUS Activity FNR::GUS GUS activity (pmol 4-MU/mgStandard Plant No. among 50 plants) Protein/min) deviation 13 1 864912974 45 2 41726 1829 14 7 23951 2443 28 9 22148 401 17 10 21557 1157 3020 13444 744 40 26 11972 1144 25 35 7662 519 35 39 5643 96 21 43 2858194 C2-(WT) 49 28 4

f) Result of Analysis of Phosphinothricin Resistance of the TransgenicTobacco Plants.

The plasmid pSUN3FNRPat was used for transformation of tobacco by usingthe Agrobacterium tumefaciens strain EHA101, as described under 3. Thetobacco plants are selectively regenerated either on phosphinothricin (5mg/l) or, as a control, on kanamycin (100 mg/l). 97% of explantsselected under kanamycin pressure (nosP:NPTII) and 40% of explantsselected under phosphinothricin pressure (FNR:Bar) developed plumulae.The plants regenerated under phosphinothricin pressure comprised boththe kanamycin and the phosphinothricin gene, demonstrating that the FNRpromoter had expressed the phosphinothricin acetyltransferase gene andthus made selection possible. Seeds of the transgenic tobacco plantswere laid out on MS medium comprising 10 mg/l phosphinothricin and therate of germination was determined. In contrast to the control ofuntransformed tobacco seeds, the seedlings developed normally. The geneof phosphinothricin acetyltransferase, which had been transferred andexpressed via the FNR promoter, was detected in the progeny of saidlines by means of PCR. The results demonstrated that the isolatednucleic acid sequence has the desired advantageous promoter properties,i.e. it shows a promoter activity which is suitable for expressingselection markers effectively and has no activity in pollen.

g) Results of the Analysis of Kanamycin Resistance of the TransgenicTobacco and Oilseed Rape Plants.

In order to study the FNR promoter-assisted imparting of resistance tokanamycin, the FNR promoter was combined with the NptII gene. Theresulting construct pS5FNRNptII was transformed into the Agrobacteriumtumefaciens strain GV3101 [mp 90] for transformation in tobacco andoilseed rape.

Seeds of the transgenic tobacco plants were laid out on MS mediumcomprising 100 mg/l kanamycin and the rate of germination wasdetermined. In contrast to the control of untransformed tobacco seeds,the seedlings developed normally. The gene of neomycinphosphotransferase (nptII), which had been transferred and expressed viathe FNR promoter, was detected in the progeny of said lines by means ofPCR.

The resulting strains have been used for transformation, as describedunder Example 2. Selective regeneration was achieved in the presence of100 mg/l (or 18 mg/l in the case of oilseed rape) kanamycin. The plantsregenerated under kanamycin pressure comprised both the kanamycin andthe phosphinothricin gene, demonstrating that the FNR promoter hadexpressed the NptII gene and thus made selection of the plants possible.

The results demonstrated that the isolated nucleic acid sequence has thedesired advantageous promoter properties, i.e. it shows a promoteractivity which is suitable for expressing selection markers effectivelyand has no activity in pollen.

Results of GUS Analysis of the Transgenic Potato Plants.

The analysis of putatively transgenic potato plants showed in 20 lines astrong GUS staining in the leaves, comparable to the expression patternof tobacco plants. With the exception of 5 plants which showed a veryweak staining in the potato tubers, no FNR promoter expression wasdetected in the remaining plants.

The data demonstrate that this promoter has very weak, if any, activityin the storage organs of potato plants and is suitable for theexpression of genes, for example of insecticides, in the leaves andother organs above the ground of plants, whose gene products areunwanted in the storage organs.

Example 5 Studies on the Suitability of the Triose PhosphateTranslocator (TPT) Promoter

Cloning of the TPT Promoter from Arabidopsis thaliana.

The putative promoter region of the TPT gene from Arabidopsis thalianawas isolated by amplification using the oligonucleotide primers L-TPTaraand R-TPTara, the ATG start codon of the TPT gene being bypassed. Usingthe primers L-TPTara and R-TPTara, the TPT promoter was amplified as a2038 bp fragment from genomic Arabidopsis thaliana DNA by means of PCR(SEQ ID NO: 3). The primer L-TPTara starts with the SalI and BamHIrestriction cleavage sites highlighted in bold type. The primer R-TPTarastarts with the AatII, SalI and XbaI restriction cleavage sites whichare located immediately upstream of the ATG start codon of the TPT geneand are highlighted in bold type.

Primer L-TPTara (SEQ ID NO: 17): 5′AAG TCG ACG GAT CCA TAA CCA AAA GAA CTC TGA TCA TGT ACG TAC CCA TT 3′Primer R-TPTara (SEQ ID NO: 18): 5′AGA CGT CGA CTC TAG ATG AAA TCG AAA TTC AGA GTT TTG ATA GTG AGA GC 3′

The TPT promoter was amplified using a “touchdown” PCR protocol with theuse of the Advantage Genomic Polymerase Mix (Clontech Laboratories, Inc;Catalogue No. #8418-1). The above-mentioned polymerase mix contains athermostable DNA polymerase from Thermus thermophilus (Tth DNApolymerase), mixed with a smaller proportion of Vent proofreading 3′-5′polymerase, and the Tth start antibody which makes hot-start PCRpossible.

Reaction Mixture:

-   -   36.8 μl H₂O    -   5 μl 10× reaction buffer (“genomic PCR”)    -   1 μl dNTP mix (10 mM each)    -   2.2 μl. 25 mM Mg(OAc).sub.2 (final concentration 1.1 mM)    -   1 μl Primer L-FNR ara (10 μM)    -   1 μl Primer R-FNR ara (10 μM)    -   1 μl 50× polymerase mix    -   2 μl Genomic Arabidopsis DNA (approx. 500 ng)

PCR Conditions:

-   -   1 cycle at 94° C. for 1 min.    -   10 cycles at 94° C. for 30 s and 70° C. for 3 min.    -   32 cycles at 94° C. for 30 s and 65° C. for 3 min.    -   1 cycle at 65° C. for 4 min.—followed by cooling to 4° C. until        further use.

b) Construction of the TPT Promoter-GUS Expression Cassette.

After gel-electrophoretic fractionation and purification from the gelusing the Quiagen PCR purification kit, the PCR product of the TPTpromoter was cloned into the pCRII vector (Invitrogen) via TA cloning.The promoter fragment was then isolated from the resulting plasmidpATTPT by means of the SalI and XbaI restriction cleavage sitesintroduced by the pair of primers and purified by gel electrophoresis.For fusion with the GUS gene, the approx. 2.0 kb TPT promoter fragmentwas cloned into the SalI/XbaI-digested binary vector pBI101. The correctinsertion of the correct fragment in the resulting plasmid pATTPT-Bi wasthen verified on the basis of a restriction analysis using theendonuclease HindIII. The plasmid pATTPT-Bi was used for transformationof tobacco.

For transformation in oilseed rape, the TPT promoter was cloned as SalIfragment of plasmid pATTPT into the vector pS3NitGUS cut with SalI andXhoI, thereby replacing the nitrilase promoter.

c) Construction of the TPT Promoter-PAT Expression Cassette.

In order to study the TPT promoter-assisted imparting of resistance tophosphinothricin, the TPT promoter was cloned as SalI fragment fromplasmid pATTPT into the SalI-cut plasmid pSUN3 PatNos upstream of thephosphinothricin resistance gene. The resulting plasmid pSUN3TPTPat wasused for transformation of tobacco using the Agrobacterium tumefaciensstrain EHA101. The tobacco plants were selectively regenerated either onphosphinothricin (5 mg/l) or, as a control, on kanamycin (100 mg/l).

d) Construction of the TPT Promoter-NptII Expression Cassette.

In order to impart resistance to kanamycin, the TPT promoter was clonedas SalI fragment into the Xha-cut dephosphorylated plasmid pSUN5NptIICat(Sungene GmbH & Co KGaA, SEQ ID NO: 24) upstream of the NPTII resistancegene. The resulting plasmid is referred to as pS5TPTNptII and wastransformed into tobacco and oilseed rape.

e) Results of GUS Analysis of the Transgenic Tobacco Plants.

Qualitative Data

Transgenic TPT::GUS-tobacco plants displayed strong GUS expression inall green tissues, especially in source leaves, here in particular inthe trichomes and the flower organs of young and fully developedflowers. GUS activity in the flower region was strongest in the ovariesand in the stigma; staining of the sepals and petals was somewhatweaker. The GUS activity was lowest in the anthers. No GUS activity wasdetected in the pollen. Transgenic oilseed rape plants showed the samestaining pattern.

In the first analysis of leaf disks of 80 in vitro-plants, 22 plantsshowed no staining whatsoever and 22 plants showed strong GUS stainingafter staining for only 3 hours. The remaining plants displayed a goodvariety of GUS stainings of various intensities in the individualplants. In the tissue culture plants, the GUS activity of the TPT::GUSplants was markedly stronger than that of the FNR::GUS plants. Seedmaterial (F1) of the lines TPT 55 and TPT 60 were analyzed with respectto their GUS activity. It turned out that strong, GUS activity wasdetected both in resting seeds and in growing seedlings (3.5 days aftersowing). In later seedling stages (6 and 10 days after sowing), thestrongest GUS activity was detected in cotyledons and in the upperregion of the seedling axis and a weaker GUS staining in the roots.Seedlings which had been cultivated in the dark displayed an unchangedGUS staining pattern.

Quantitative Data

Quantitative analysis of the GUS activity in TPT::GUS transgenic tobaccoplants (transformed with plasmid pAT-TPT-Bi) was carried out on thefirst fully developed leaves of tobacco plants 19 days after transferfrom the tissue culture to the greenhouse. The data correspond to theaverage of four independent measurements.

TABLE 5 Quantification of GUS activity in leaf tissue of selectedtransgenic TPT::GUS tobacco plants (transformed with plasmidpAT-TPT-Bi). WT2: controls from untransformed wild-type plants. Rank(x-strongest GUS Activity TPT::GUS GUS activity (pmol 4-MU/mg StandardPlant no. among 50 plants) Protein/min) deviation 55  1st 62910 3576 15  2nd 58251 2533 10  5th 36759 1008 60 10th 19536 1783 56 11th 188761177 43 12th 18858 1404 27 35th 7390 233 44 59th 311 24 80-WT2 80th 5 13

Results of GUS Analysis of the Transgenic Potato Plants.

The analysis of putatively transgenic potato plants showed in 28 linesstrong GUS staining in the leaves, comparable to the expression patternof tobacco plants. A strong staining was likewise detected in the potatotubers of the transgenic plants. This demonstrates that the TPT promoteris expressed strongly and ubiquitously in potatoes, too.

g) Results of the Analysis of Phosphinothricin Resistance of theTransgenic Tobacco Plants.

The plasmid pSUN3TPTPat was used for transformation of tobacco by usingthe Agrobacterium tumefaciens strain EHA101, as described under 3. Thetobacco plants are selectively regenerated either on phosphinothricin (5mg/l) or, as a control, on kanamycin (100 mg/l). 97% of explantsselected under kanamycin pressure and 70% of explants selected underphosphinothricin pressure developed plumulae. The plants regeneratedunder phosphinothricin pressure comprised both the kanamycin and thephosphinothricin gene, demonstrating that the TPT promoter had expressedthe phosphinothricin acetyltransferase gene and thus made selectionpossible. Seeds of the transgenic tobacco plants were laid out on MSmedium comprising 10 mg/l phosphinothricin and the rate of germinationwas determined. In contrast to the control of untransformed tobaccoseeds, the seedlings developed normally. The gene of phosphinothricinacetyltransferase, which had been transferred and expressed via the TPTpromoter, was detected in the progeny of said lines by means of PCR. Theresults demonstrate that the isolated nucleic acid sequence has thedesired advantageous promoter properties, i.e. it shows a promoteractivity which is suitable for expressing selection markers effectivelyand has no activity in pollen.

h) Results of the Analysis of Kanamycin Resistance of the TransgenicTobacco and Oilseed Rape Plants.

In order to study the TPT promoter-assisted imparting of resistance tokanamycin, the TPT promoter was combined with the NptII gene. Theresulting construct pS5TPTNptII was transformed into the Agrobacteriumtumefaciens strain GV3101 [mp 90] for transformation in tobacco andoilseed rape.

The resulting strains have been used for transformation, as describedunder Example 2. Selective regeneration was achieved in the presence of100 mg/l (or 18 mg/l in the case of oilseed rape) kanamycin. The plantsregenerated under kanamycin pressure comprised both the kanamycin andthe phosphinothricin gene, demonstrating that the TPT promoter hadexpressed the NptII gene and thus made selection of the plants possible.

Seeds of the transgenic tobacco plants were laid out on MS mediumcomprising 100 mg/l kanamycin and the rate of germination wasdetermined. In contrast to the control of untransformed tobacco seeds,the seedlings developed normally. The gene of neomycinphosphotransferase (nptII), which had been transferred and expressed viathe TPT promoter, was detected in the progeny of said lines by means ofPCR.

The results demonstrate that the isolated nucleic acid sequence has thedesired advantageous promoter properties, i.e. it shows a promoteractivity which is suitable for expressing selection markers effectivelyand has no activity in pollen.

i) Cloning of the Truncated TPT Promoter (STPT).

The truncated putative promoter region of the Arabidopsis thaliana TPTgene (STPT) was isolated from the plasmid pATTPT (SEQ ID NO: 27) byamplification by means of PCR using the primer 5-TPTara (SEQ-ID NO: 26)and R-TPTara (see above SEQ ID NO: 18).

Reaction Mixture:

-   -   37.8 μl H₂O    -   5 μl 10× Reaction buffer (“genomic PCR”)    -   1 μl dNTP mix (2.5 mM each)    -   2.2 μl 25 mM Mg(OAc).sub.2 (final concentration 1.1 mM)    -   1 μl Primer 5-TPTara (10 μM)    -   1 μl Primer R-TPTara (10 μM)    -   1 μl 50× polymerase mix (“Advantage Genomic Polymerase Mix”;        Clontech Lab., Inc.; Cat.-No.: #8418-1)    -   1 μpATTPT plasmid DNA (1 ng)

PCR Conditions:

-   -   1 cycle at 94° C. for 5 min    -   25 cycles at 94° C. for 30 s and 52° C. for 1 min.    -   1 cycle at 52° C. for 4 min., followed by cooling to 4° C. until        further use.

Promoter Primer 5-TPTara (SEQ ID NO: 26)5′-AAG TCG ACG GAT CCT-GAT-AGC-TTA-TAC-TCA-AAT- TCA-ACA-AGT-TAT-3′

The 1.3 kb PCR product of the truncated TPT promoter was cloned, aftergel-electrophoretic fractionation and purification from the gel, intothe SmaI-cut and dephosphorylated vector pUC18, using the SureCloneLigation Kit (Amersham Pharmacia Biotech; Code-No.: 27-9300-01). Theresulting plasmid is referred to as pATSTPT. The sequence was checked bymeans of sequencing.

j) Construction of the STPT Promoter-NptII Expression Cassette.

In order to impart resistance to kanamycin, the STPT promoter (SEQ IDNO: 27) was cloned as SalI fragment into the XhoI-cut dephosphorylatedplasmid pSUN5NptIICat (Sungene GmbH & Co KGaA, SEQ ID NO: 24) upstreamof the NptII resistance gene. The resulting plasmid is referred to aspS5STPTNptII and was transformed into tobacco and oilseed rape.

k) Results of the Analysis of Kanamycin Resistance of the TransgenicTobacco and Oilseed Rape Plants.

In order to study the STPT promoter-assisted imparting of resistance tokanamycin, the plasmid pS5STPTNptII was transformed into theAgrobacterium tumefaciens strain GV3101 [mp 90] for transformation intotobacco and oilseed rape. The resulting strain has been used fortransformation, as described under Example 2. Selective regeneration wasachieved in the presence of 100 mg/l (or 18 mg/l for oilseed rape)kanamycin.

The results demonstrate that the isolated nucleic acid sequence has thedesired advantageous promoter properties, i.e. it shows a promoteractivity which is suitable for expressing selection markers effectively.

Example 6 Comparison of the Transformation Efficiencies of the FNR andTPT Promoters and of the NOS Promoter

In a comparative experiment, the efficiency of the transformation oftobacco was determined using the FNR promoter (FNR-P), the TPT promoter(TPT-P) and the Nos promoter (Nos-P). The promoters were, as described,fused in each case to the NptII gene. After the plumulae had formed and,respectively, the shoot had roots on kanamycin-comprising medium, theresistant transformants were counted and their numbers were compared. APCR which was used to detect the NptII gene showed the high proportionof transgenic plants.

TABLE 6 Transformation efficiency NOS-P FNR-P TPT-P Shoot formation100%  68% 76% Rooted plants 80% 81% 80% Transgenic plants 92% 100% 100% 

Comparative Example 1 Studies of the Suitability of the UbiquitinPromoter

a) Cloning of the Ubiquitin Promoter from Arabidopsis thaliana.

The ubiquitin promoter was amplified from genomic Arabidopsis thalianaDNA by means of PCR using the primers ubi5 and ubi3.

ubi5 (SEQ ID NO: 19): 5′-CCAAACCATGGTAAGTTTGTCTAAAGCTTA-3-′ubi3 (SEQ ID NO: 20): 5′-CGGATCCTTTTGTGTTTCGTCT-TCTCTCACG-3′

Reaction Mixture:

-   -   37.5 μl H₂O    -   5 μl 10× reaction buffer (“genomic PCR”)    -   4 μl dNTP mix (2.5 mM each)    -   2.2 μl 25 mM Mg(OAc)₂ (final concentration 1.1 mM)    -   1 μl Primer ubi3 (10 μM)    -   1 μl Primer ubi5 (10 μM)    -   0.5 μl Pfu-turbo polymerase mix    -   1 μl Genomic Arabidopsis DNA (ca. 250 ng)

PCR Conditions:

-   -   1 cycle at 94° C. for 5 min.    -   25 cycles at 94° C. for 30 s, 52° C. for 1 min. and 72° C. for 1        min.    -   1 cycle at 52° C. for 1 min. and 72° C. for 10 min, followed by        cooling to 4° C. until further use.

The resultant PCR fragment was cooled as HindIII/BamHI fragment into theHindIII/BamHI-cut plasmid pGUSINT37 (pUBI42GUS) and verified by means ofsequence analysis.

b) Cloning of the Ubiquitin Promoter Upstream of the PAT Gene.

In order to study the ubiquitin promoter-assisted imparting ofresistance to phosphinothricin, the ubiquitin promoter was cloned asBamHIII/HindIII fragment into the BamHI/HindIII-cut plasmid pSUN3 PatNosupstream of the phosphinothricin resistance gene. The resulting plasmidpSUN3UBIPat was used for transformation of tobacco using theAgrobacterium tumefaciens strain EHA101. The tobacco plants wereselectively regenerated either on phosphinothricin (5 mg/l) or, as acontrol, on kanamycin (100 mg/l).

c) Results of the Analysis of Phosphinothricin Resistance of theTransgenic Tobacco Plants.

In contrast to selection on kanamycin, which was normal, no calli orshoots were obtained under selection on phosphinothricin. Thus, theubiquitin promoter is unsuitable for expression of a selective markerfor the Agrobacterium tumefaciens-mediated gene transfer with subsequentregeneration of tissues.

Comparative Example 2 Studies on the Suitability of the SqualeneSynthase (SQS) Promoter

a) Cloning of the Squalene Synthase (SQS) Promoter from Arabidopsisthaliana

The squalene synthase promoter was amplified from genomic Arabidopsisthaliana DNA by means of PCR using the primers sqs5 and sqs3.

sqs5 (SEQ ID NO: 21): 5′-GTCTAGAGGCAAACCACCGAGTGTT-3′sqs3 (SEQ ID NO: 22): 5′-CGGTACCTGTTTCCAGAAAAT-TTTGATTCAG-3′

Reaction Mixture:

-   -   37.5 μl H₂O    -   5 μl 10× Reaction buffer (“genomic PCR”)    -   4 μl dNTP mix (2.5 mM each)    -   2.2 ml 25 mM Mg(OAc)₂ (final concentration 1.1 mM)    -   1 μl Primer sqs3 (10 μM) (10 μM)    -   1 μl Primer sqs5 (10 μM)    -   0.5 μl Pfu-turbo polymerase mix    -   1 μl Genomic Arabidopsis DNA (approx. 250 ng)

PCR Conditions:

-   -   1 cycle at 94° C. for 5 min.    -   25 cycles at 94° C. for 30 s, 52° C. for 1 min. and 72° C. for 1        min.    -   1 cycle at 52° C. for 1 min. and 72° C. for 10 min., followed by        cooling to 4° C. until further use.

The resultant PCR fragment was cloned as XbaII/BamHI fragment into theXbaII/BamHI-cut plasmid pGUSINT37 (PSQSPGUS) and verified by means ofsequence analysis.

b) Cloning of the Squalene Synthase Promoter Upstream of the PAT Gene.

In order to study the squalene synthase promoter-assisted imparting ofresistance to phosphinothricin, the squalene synthase promoter wascloned as BamHI/SalI fragment into the BamHI/SaliI-cut plasmid pSUN3PatNos upstream of the phosphinothricin resistance gene. The resultingplasmid pSUN3SQSPat was used for transformation of tobacco using theAgrobacterium tumefaciens strain EHA101. The tobacco plants wereselectively regenerated either on phosphinothricin (5 mg/l) or, as acontrol, on kanamycin (100 mg/l).

c) Results of the Analysis of Phosphinothricin Resistance of theTransgenic Tobacco Plants.

In contrast to selection on kanamycin, which was normal, no calli orshoots were obtained under selection on phosphinothricin. Thus, theubiquitin promoter is unsuitable for expression of a selective markerfor the Agrobacterium tumefaciens-mediated gene transfer with subsequentregeneration of tissues.

Comparative Example 3 Promoter Activity Assay of the Ubiquitin andSqualene Synthase-Promoter by Means of a Particle Gun

In order to assay the activity of the ubiquitin promoter and thesqualene synthase promoter, sterile tobacco leaves were bombarded withplasmid DNA of plasmids pUBI42GUS, pSQSPGUS and pGUSINT37 by means ofthe BioRad Biolistics particle gun. In this connection, microcarriers(25 μg of Gold, Heraeus 0.3 to 3 μm) were treated with 10 μg of plasmidDNA, 2.5 M CaCl₂, and 0.1 M spermidine, washed with alcohol and fired atthe leaves which were lying on MS agar medium under a vacuum of 26inches and a pressure of 1100 psi. The explants were then incubated inMS medium comprising 2% sucrose for 24 h and then histochemicallystained with X-gluc.

In contrast to the comparative construct pGUSINT37 in which the GUS genewas expressed under the control of the 35S promoter, the ubiquitinpromoter and the squalene synthase promoter showed only very few andvery weak GUS-stained dots. This indicates that the ubiquitin andsqualene synthase promoter activities are distinctly weaker than theCaMV35S promoter activity.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all U.S. and foreign patents and patent applications, and allpublications or other documentary materials, are specifically andentirely hereby incorporated herein by reference. It is intended thatthe specification and examples be considered exemplary only, with thetrue scope and spirit of the invention indicated by the followingclaims.

1. An expression cassette for transgenic expression of nucleic acids,comprising: a promoter containing the sequence of SEQ ID NO: 1 or 2, ora fragment, functional equivalent or equivalent fragment thereof thatpossesses a promoter activity, wherein said promoter or said fragment isfunctionally linked to a nucleic acid sequence to be expressedtransgenically.
 2. The expression cassette of claim 1, wherein theequivalent fragment comprises the sequence of SEQ ID NO:
 4. 3. Theexpression cassette of claim 1, wherein: the nucleic acid sequence to beexpressed is functionally linked to one or more further genetic controlsequences, or the expression cassette contains one or more additionalfunctional elements.
 4. The expression cassette of claim 1, wherein thenucleic acid sequence to be expressed transgenically enables: expressionof a protein encoded by said nucleic acid sequence, or expression of asense or antisense RNA or ribozyme encoded by said nucleic acidsequence.
 5. The expression cassette of claim 1, wherein the nucleicacid sequence to be expressed transgenically is selected from the groupconsisting of nucleic acids coding for selection markers, reportergenes, cellulases, chitinases, glucanases, ribosome-inactivatingproteins, lysozymes, Bacillus thuringiensis endotoxin, α-amylaseinhibitor, protease inhibitors, lectins, RNAases, ribozymes, acetyl-CoAcarboxylases, phytases, 2S albumin from Bertholletia excelsa, antifreezeproteins, trehalose phosphate synthase, trehalose phosphate phosphatase,trehalase, DREB1A factor, farnesyl transferases, ferritin, oxalateoxidase, calcium-dependent protein kinases, calcineurins, glutamatedehydrogenases, N-hydroxylating multifunctional cytochrome P450,transcriptional activator CBF1, phytoene desaturases,polygalacturonases, flavonoid 3′-hydroxylases, dihydroflavanol4-reductases, chalcone isomerases, chalcone synthases, flavanone3-beta-hydroxylases, flavone synthase II, branching enzyme Q, starchbranching enzyme, and combinations thereof.
 6. The expression cassetteof claim 1, wherein the nucleic acid sequence to be expressedtransgenically is selected from the group consisting of the nucleic acidsequences with GenBank accession numbers U77378, AF306348, A19451,L25042, S78423, U32624, X78815, AJ002399, AF078796, ABO44391, AJ222980,X14074, AB045593, AF017451, AF276302, ABO61022, X72592, AB045592, andAR123356.
 7. The expression cassette of claim 1, wherein the nucleicacid sequence to be expressed transgenically is selected from the groupconsisting of positive selection markers, negative selection markers,factors that provide a growth advantage and combinations thereof.
 8. Theexpression cassette of claim 7, wherein the positive or negativeselection marker is selected from the group consisting of proteins thatimpart a resistance to antibiotics, metabolism inhibitors, herbicides,biocides and combinations thereof.
 9. The expression cassette of claim7, wherein the positive or negative selection marker is selected fromthe group consisting of proteins that impart a resistance tophosphinothricin, glyphosate, bromoxynil, dalapon, 2-deoxyglucose6-phosphate, tetracyclines, ampicillin, kanamycin, G418, neomycin,paromomycin, bleomycin, zeocin, hygromycin, chloramphenicol, sulfonylurea herbicides, imidazolinone herbicides and combinations thereof. 10.The transgenic expression cassette of claim 7, wherein the selectionmarker is selected from the group consisting of phosphinothricinacetyltransferases, 5-enolpyruvylshikimate 3-phosphate synthases,glyphosate oxidoreductases, dehalogenases, nitrilases, neomycinphosphotransferases, DOG^(R)1 genes, acetolactate synthases, hygromycinphosphotransferases, chloramphenicol acetyltransferases, streptomycinadenylyltransferases, β-lactamases, tetA genes, tetR genes, isopentenyltransferases, thymidine kinases, diphtheria toxin A, cytosine deaminase(codA), cytochrome P450, haloalkanedehalogenases, iaaH gene, tms2 gene,β-glucuronidases, mannose 6-phosphate isomerases, UDP-galactose4-epimerases and combinations thereof.
 11. The transgenic expressioncassette of claim 7, wherein the positive or negative selection markeris encoded by a nucleic acid that contains: the sequence of SEQ ID NO: 5or 6; or the sequence of GenBank Acc. No.: X17220, X05822, M22827,X65195, AJ028212, X17220, X05822, M22827, X65195, AJ028212, X63374,M10947, AX022822, AX022820, E01313, J03196, AF080390, AF234316,AF080389, AF234315, AF234314, U00004, NC001140, X51514, AB049823,AF094326, X07645, X07644, A19547, A19546, A19545, 105376, 105373,X74325, AF294981, AF234301, AF234300, AF234299, AF234298, AF354046,AF354045, X65876, X51366, AJ278607, L36849, AB025109, or AL133315.
 12. Avector comprising the expression cassette of claim
 1. 13. A transgenicorganism transformed with the vector of claim
 12. 14. The transgenicorganism of claim 13, which is selected from the group of organismsconsisting of bacteria, yeasts, fungi, animal, and plant organisms. 15.The transgenic organism of claim 13, which is selected from the group oforganisms consisting of Arabidopsis, tomato, tobacco, potatoes, corn,oilseed rape, wheat, barley, sunflowers, millet, beet, rye, oats,sugarbeet, bean plants and soyabean.
 16. A cell culture, plant ortransgenic propagation material, derived from the transgenic organism ofclaim
 13. 17. A method for transgenic expression of nucleic acidscomprising: transgenically expressing a nucleic acid sequence that isfunctionally linked to: a promoter containing the sequence of SEQ ID NO:1 or 2; or a fragment, functional equivalent or equivalent fragmentthereof that possesses a promoter activity.
 18. The method of claim 17,wherein the functionally equivalent fragment comprises the sequence ofSEQ ID NO:
 4. 19. The method of claim 17, wherein the nucleic acidsequence to be expressed is functionally linked to one or more furthergenetic control sequences, and one or more additional functionalelements.
 20. The method of claim 17, wherein: the nucleic acid sequenceto be expressed is functionally linked to one or more further geneticcontrol sequences; or one or more additional functional elements. 21.The method of claim 17, wherein the nucleic acid sequence to beexpressed transgenically enables: expression of a protein encoded bysaid nucleic acid sequence, or expression of a sense or antisense RNA orribozyme encoded by said nucleic acid sequence.
 22. The method of claim17, wherein the nucleic acid sequence to be expressed transgenically isselected from the group consisting of nucleic acids coding for selectionmarkers, reporter genes, cellulases, chitinases, glucanases,ribosome-inactivating proteins, lysozymes, Bacillus thuringiensisendotoxin, α-amylase inhibitor, protease inhibitors, lectins, RNAases,ribozymes, acetyl-CoA carboxylases, phytases, 2S albumin fromBertholletia excelsa, antifreeze proteins, trehalose phosphate synthase,trehalose phosphate phosphatase, trehalase, DREB1A factor, farnesyltransferases, ferritin, oxalate oxidase, calcium-dependent proteinkinases, calcineurins, glutamate dehydrogenases, N-hydroxylatingmultifunctional cytochrome P450, transcriptional activator CBF1,phytoene desaturases, polygalacturonases, flavonoid 3′-hydroxylases,dihydroflavanol 4-reductases, chalcone isomerases, chalcone synthases,flavanone 3-beta-hydroxylases, flavone synthase II, branching enzyme Q,starch branching enzyme, and combinations thereof.
 23. The method ofclaim 17, wherein the nucleic acid sequence to be expressedtransgenically is selected from the group consisting of the nucleic acidsequences with GenBank accession numbers U77378, AF306348, A19451,L25042, S78423, U32624, X78815, AJ002399, AF078796, AB044391, AJ222980,X14074, AB045593, AF017451, AF276302, AB061022, X72592, AB045592, andAR123356.
 24. A method for selecting transformed organisms comprising:introducing a nucleic acid sequence coding for a selection marker to anorganism, wherein said nucleic acid sequence is functionally andtransgenically linked to a promoter comprising the sequence of SEQ IDNO: 1 or 2; or a fragment, functional equivalent or equivalent fragmentthereof that possesses a promoter activity; selecting organismsexpressing said selection marker; and isolating selected organisms. 25.The method of claim 22, wherein the selection marker is selected fromthe group consisting of the nucleic acid sequences with GenBankaccession numbers U77378, AF306348, A19451, L25042, S78423, U32624,X78815, AJ002399, AF078796, ABO44391, AJ222980, X14074, AB045593,AF017451, AF276302, ABO61022, X72592, AB045592, and AR123356.
 26. Themethod of claim 22, wherein the selection marker is selected from thegroup consisting of positive selection markers, negative selectionmarkers, factors that provide a growth advantage, proteins that impart aresistance to antibiotics, metabolism inhibitors, herbicides, biocides,phosphinothricin acetyltransferases, 5-enolpyruvylshikimate 3-phosphatesynthases, glyphosate oxidoreductases, dehalogenases, nitrilases,neomycin phosphotransferases, DOG^(R)1 genes, acetolactate synthases,hygromycin phosphotransferases, chloramphenicol acetyltransferases,streptomycin adenylyltransferases, β-lactamases, tetA genes, tetR genes,isopentenyl transferases, thymidine kinases, diphtheria toxin A,cytosine deaminase (codA), cytochrome P450, haloalkanedehalogenases,iaaH gene, tms2 gene, β-glucuronidases, mannose 6-phosphate isomerases,UDP-galactose 4-epimerases and combinations thereof.
 27. A method forthe production of a foodstuff, a feedstuff, a seed, a pharmaceutical ora fine chemical comprising: providing a transgenic organism containingan expression cassette for transgenic expression of nucleic acids thatcomprise a promoter containing the sequence of SEQ ID NO: 1 or 2, or afragment, functional equivalent or equivalent fragment thereof thatpossesses a promoter activity, wherein said promoter or said fragment isfunctionally linked to a nucleic acid sequence to be expressedtransgenically; and propagating said transgenic organism or cellcultures, parts or transgenic propagation material derived therefrom.28. The method of claim 27, which further comprises isolating thefoodstuff, feedstuff, seed, pharmaceutical or fine chemical.
 29. Themethod of claim 28, wherein the pharmaceutical is an antibody, enzyme,pharmaceutically active protein or combination thereof.
 30. The methodof claim 28, wherein the fine chemical is selected from the groupconsisting of enzymes, vitamins, amino acids, sugars, saturated orunsaturated fatty acids, natural or synthetic flavorings, aromatizingsubstances, and colorants.
 31. A plant cell, plant or part thereof, orpropagation material obtained therefrom, which plant cell, plant or partthereof, or propagation material derived therefrom comprises theexpression cassette of claim 1.